\r\n\tOne of the most important elements that contributes to fatigue failures are the welded joints due to different factors that act over the joint and are producing early failure. Fatigue cracks usually initiate from surface of the component and propagate across the transverse section, perpendicular to the stress direction. Consequently, the crack propagation and fracture mechanics theories should be studied if we want to understand and to find the causes that produce the component failures and to calculate the residual life. Another factor that affects the crack propagation and fatigue is the environment. A comprehensive understanding of the influence of environment over the crack propagation and fatigue has been blocked by the complexity of the problem. The difficulties in understanding the various micromechanisms governing crack initiation and crack propagation are caused by a lack of a truly interdisciplinary approach to the problem. \r\n\tFrom another side, currently, numerical models and simulation have become a good alternative to solve problems related to fatigue and crack propagation due the relatively low cost and short time expended in this labor, so the book will try to give an overview of the topic. Generally, the fatigue has been studied through the materials more used in engineering (steel, aluminum, bronze), but in recent years, the use of the non-conventional materials like naturals (bamboo, wood, natural fibers), composite materials and others has taken great relevance in engineering applications.
",isbn:"978-1-78985-994-2",printIsbn:"978-1-78985-993-5",pdfIsbn:null,doi:null,price:0,priceEur:0,priceUsd:0,slug:null,numberOfPages:0,isOpenForSubmission:!1,hash:"1ec891200aebef4dc2b19461e0ee4068",bookSignature:"Ph.D. Hector Enrique Jaramillo S. and Dr. Julian Arnaldo Avila",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/9265.jpg",keywords:"Finite Element Analysis, Fracture, Residual Stress, Stability , Mechanical Strength , Biomechanics Behavior , Strain Energy Functions, Yield Strength , Tensile Strength, Convergence, Composite Materials, Biologic Materials",numberOfDownloads:309,numberOfWosCitations:0,numberOfCrossrefCitations:0,numberOfDimensionsCitations:0,numberOfTotalCitations:0,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"August 1st 2019",dateEndSecondStepPublish:"August 22nd 2019",dateEndThirdStepPublish:"October 21st 2019",dateEndFourthStepPublish:"January 9th 2020",dateEndFifthStepPublish:"March 9th 2020",remainingDaysToSecondStep:"4 months",secondStepPassed:!0,currentStepOfPublishingProcess:4,editedByType:null,kuFlag:!1,editors:[{id:"255849",title:"Ph.D.",name:"Hector",middleName:"Enrique",surname:"Jaramillo S.",slug:"hector-jaramillo-s.",fullName:"Hector Jaramillo S.",profilePictureURL:"https://mts.intechopen.com/storage/users/255849/images/system/255849.jpeg",biography:"Héctor E. Jaramillo S. is a full-time professor-researcher of the Autonoma de Occidente University (Cali, Colombia, Sour America). He has a Ph.D. of the Universidad del Valle with emphasis in mechanical of solids, the Ph.D. dissertation was about the design and development of a finite element model of the L4-L5-S1 segment of the human spine. In order to calibrate the model, Prof. Jaramillo had to perform the experimental characterization of the segment in the Orthopaedic Bioengineering Research Laboratory of the Colorado State University. He has a lot of experience in finite element analysis using nonlinear, ortotropic, hiperelastic and particularly non-conventional materials as biologic materials. He has a master’s degree in Civil Engineering (2004), and B.Sc. in Mechanical Engineering (1992). 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1. Introduction
Turkey is one of the richest countries in variability of flora. It has nearly 9000 plant species about 3000 of which are endemic [1]. Asteraceae, is represented by 50 species in Turkey with an endemism of nearly 54% [2]. Rhaponticoides mykalea (Hub.-Mor.) M.V. Agab. & Greuter which belongs to the Asteraceae family, falls within the CR (Critically Endangered) category in the Red Data Book of Turkey [1]. While R. mykalea (Hub.-Mor.) was classified under the section Centaurea as Centaurea mykalea (Hub.-Mor.) before now. Today it has been separated from the section Centaurea [3]. It spreads very scarce in Kuşadası (Aydın), Muğla and Isparta, and faces with the danger of extinction. R. mykalea that has very limited number of individuals is under strong anthropogenic pressure such as the gradually increase in ongoing urbanization due to rapid developments of tourism sector, the conversion of natural habitats into human dominated lands, the over-grazing and collecting capitula of R. mykalea by local people for food. The species has already been under the threat of extinction and the situation above will increase the risk of extinction of this species even more [4]. For this reason, local protection measures and global conservation strategies are necessary [5].
Nowadays, the conservation of wild plant genetic resources is very important for preventing a decrease in genetic variability. Conservation of the endemic or threatened plants is carried out using different strategies. In vitro culture is an efficient method for ex situ conservation of plant diversity [6,7], because many endangered species can be quickly propagated and preserved from a minimum of plant material with low impact on wild populations with this technology [8]. In recent years, there has been an increased interest in in vitro techniques that offer powerful tools for germplasm conservation and the mass multiplication of many threatened plant species [9]. Especially in vitro propagation of endangered plants can offer considerable benefits for the rapid cultivation of at risk species that have a limited reproductive capacity and exist in threatened habitats [5].
Micropropagation constitutes a powerful tool for ex situ conservation programs of threatened plants, especially for species with very reduced populations or low seed production [6,7]. This technique facilitates the rapid establishment of a large number of stock plants, from a minimum of original plant material, thus imposing minimum impact on the endangered wild populations. Axillary shoot proliferation typically results in average tenfold increase in shoot number per monthly culture passage. In a period of 6 months, it is feasible to obtain as many as 1 000 000 propagulesor plants, starting from a single explant [10].With this technology various endemic and endangered species have been successfully propagated; such as and Centaurea paui [8], Anthemis xylopoda O.Schwarz [11], Centaurea spachii [12], Centaurea zeybekii [13], Centaurea junoniana [14], Astragalus chrysochlorus [15], Centaurium rigualii [16] and Syzygium alternifolium [17].
However, during our literature search, no report concerning in vitro regeneration of R.mykalea by axillary shoot proliferation was found.
The objective of the present study was to establish an efficient in vitro method for the rapid propagation via axillary shoot propagation of R.mykalea, a critically endangared endemic plant species. The shoots that were obtained from in vitro germinated mature embryos were used for axillary shoot proliferation. For that reason, the most appropriate cytokinin type and concentration were determined.
2. Material and methods
2.1. Plant material and explant source
Capitula of Rhaponticoides mykalea were collected from a wild population in Aydın-Turkey (Samsun mountain, localities: N 37 º 47.01 “ ; E 027º 19.16 “) during summer period (July and August -2008) before seed dormancy period (Figure 1).
R. mykalea has been propagated from seed in the past [18]. However, researchers have explained that the seed is not suitable explant for in vitro propagation of R. mykalea due to strong seed dormancy and low germination frequency even after dormancy period. Therefore, embryos isolated from achenes which have not yet crossed dormancy periods were used as initial explant.
The achenes isolated from capitula were sterilised, and mature zygotic embryos that were dissected out from achene were used as initial explant. Mature zygotic embryos were dissected out from achenes and cultured on Murashige and Skoog (MS) [19] basal medium for germination. The shoots that were obtained from in vitro germinated mature embryos were used for axillary shoot proliferation.
2.2. Achene viability
Achene viability was subjected to tetrazolium test.Tetrazolium test is based on reduction of colourless solution 2,3,5–tripheniltetrazolim chloride or bromide into insoluble 2,3,5– triphenilformazan red in colour. This solution acts as an indicator for detection of reduction processes that take place in living parts of the seed. Inside the seed, tetrazolium intakes hydrogen from dehydrogenase. By hidrogenization of tetrazolium a red, stable substance called formazan, which dyes living parts of the seed, is formed in the living cells.Tissue of many plant species must be removed to introduce the dye into the tissue. Tissue removal can be done by pilling the seed coat off, punching, and longitudinal or cross-cutting of unessential seed parts. Prepared seed is submerged into 0,5 – 1% tetrazolium solution. Seed must be completely covered with solution, and not exposed to direct light. After the time needed for dyeing expires (it depends on plant species) the estimation of dyeing is approached. All tissue (necessary for normal seedling development) of a viable seed should be dyed. Except completely dyed, viable seeds, and completely undyed, unviable seeds, a partly dyed seeds may also be found. Depending on the species, small undyed spots of some parts of these tissues may be accepted. Location, size of undyed areas, and sometimes intensity of dyeing, determine whether some seed is considered as viable or not [20].
To determine achene viability of R. mykalea, longitudinally-halved seeds were treated in tetrazolium solution (TTC, 1%) for 2 h at room temperature. After that time, red staining embryos were evaluated as alive.
2.3. Seed sterilisation, media preparation and culture conditions
In order to determine proper sterilisation procedure, achenes isolated from capitula were washed thoroughly under running tap water for 30 mins. Subsequently at various times, achenes were put in 70% (w/v) ethanol and 4.5% (w/v) sodium hypochlorite containing 2 drops of wetting agent (Tween-80); afterwards, the achenes were rinsed three times (5 mins each) with sterile distilled water in a laminar flow hood. After sterilisation, zygotic embryos were isolated from achenes and cultured on PDA (Potato Dextrose Agar) to determine early contamination. PDA cultures were maintained at 24 ± 2 ºC for 3 days. At the end of this period, observations were made in order to determine the appropriate sterilisation time.
All the experiments were maintained on semi-solid basal medium supplemented with or without various concentration of plant growth regulators. Basal medium contained Murashige and Skoog (MS) [19] mineral salts, 100 mgl-1 myo-inositol, 2 mgl-1 glycine, 0.5 mgl-1 nicotinic acid, 0.5 mgl-1 pyridoxine-HCl, 0.1 mgl-1 thiamine-HCL, 3% (w/v) sucrose, 8 gl-1 agar (Agar-agar), various concentration of plant growth regulators 1N6- Benzyladenine (BA) and Kinetin (KIN), indole-3-butyric acid (IBA), indole-3-acetic acid (IAA) or naphthalene acetic acid (NAA) were used in experiments depending on experimental objectives.
The pH of media was adjusted to 5.8 with 1M NaOH or HCl prior to autoclaving at 105 kPa and 121° C for 15 min. Culture vessels were 190 ml glass jars containing 30 ml of medium.
2.4. Axillary shoot proliferation
Mature embryos that were isolated from achenes were cultured on MS basal medium to obtain sterile seedlings (unpublished data). After eight weeks, seedlings (~2-3 cm), were separated from primary roots and transferred to MS medium containing different concentrations of BA or KIN (0.1, 0.5, 1.0 and 2.0 mgl-1) for axillary shoot propagation. A control treatment without cytokinins was also included. At the end of the 3 subculture, the number of shoots per explant and average shoot length was evaluated for each cytokinin type and concentration.
Axillary shoot proliferation experiments were conducted with 15 replications consisting of one explant per jar and were repeated three times. Cultures were incubated at 24 ± 2 °C under a light regime of 16 h photoperiod by cool-white fluorescent lamps. The cultures were subcultured to fresh medium of the same composition at an interval of 4 weeks.
2.5. Shoot rooting and acclimatization of plantlets
After three subcultures, elongated shoots (~4 cm) were excised stock cultures and transferred to MS and half strength MS medium (½ MS) with or without different concentrations (0.5, 1.0, 2.0 and 5.0 mgl-1) of auxins (IBA, IAA or NAA) for rooting. The results of rooting experiments were expressed in percentage after 6 weeks of culture initiation.
Rooting experiments were conducted with 15 replications consisting of one explant per jar and were repeated three times. Cultures were incubated at 24 ± 2 °C under a light regime of 16 h photoperiod by cool-white fluorescent lamps. The cultures were subcultured to fresh medium of the same composition at an interval of 4 weeks.
After 8 weeks of rooting in vitro, the plantlets were removed from the culture jars then the agar was carefully washed off the rooted plantlets to minimize pathogen attack. The plantlets were planted into 10 cm diameter plastic pots containing garden soil and kept in the growth chamber under 24 ± 2°C and 16-h light photoperiod. After 4 weeks the plantlets kept at normal laboratory conditions.
2.6. Statistical analyses
Means of shoot number per explant, shoot lenght and frequency of rooting were analyzed by one-way analysis of variance (ANOVA, SPSS for Windows v.9., SPSS, USA). Differences were analyzed by analysis of variance and the means compared using Duncan’s multiple range test at p< 0.05. Data giving in percentages were subjected to x´ = arcsine √(x/100) transformation [21].
3. Results and discussion
The viability percentage of achenes was 80% according to Tetrazolium test. According to our results, the most suitable sterilisation procedure of achenes is as follows: The achenes are washed thoroughly under running tap water for 30 mins. After this process, seeds must be exposed to 70% (w/v) ethanol for five mins and then to 4.5% (w/v) sodium hypochlorite containing 2 drops wetting agent (Tween-80) for eight mins. Finally, seeds are rinsed three times with sterilised distilled water (5 mins each). Sterile cultures are than obtained in high proportion (100%).
Four-week-old sterile seedlings obtained from mature zygotic embryos were used as explant for axillary shoot proliferation experiments. At the end of the experiments, the most appropriate cytokinin type and concentration were determined (Figure 2). Axillary shoot propagation of R. mykalea was obtained in all media without or with cytokinin. Cytokinins are generally recognized as critical for the production of shoot primordia under in vitro conditions. Both cytokinins induced healthy shoots in our study. However, it is shown that BA is more effective cytokinin than KIN. The maximum shoot number per explant were obtained in 0.5 mgl-1 BA added MS medium (5.8 shoot/explant) (Figure 2 and 3).
A decrease in the number of shoots were observed at both higher (1 and 2 mgl-1) and lower concentrations of BA (0.1 mgl-1). Similar results were also reported for axillary shoot proliferation of Centaurea spachii [12] and Centaurea zeybekii [13]. BA was also reported as an effective cytokinin for other endemic and threatened Centaurea species [14, 16]. However, BA was evaulated as an effective cytokinin for shoot multiplication in many species of Asteraceae; such as Centaurea junoniana [14], Gerbera jamesonii hybrida [22], Centaurium rigualii [16], Syzygium alternifolium [17] and Anthemis xylopoda [11].
MS medium supplemented with 0.1 mgl-1 Kinetin (KIN) was determined as the most suitable medium for the maximum shoot length (7.35 cm) (Figure 4). While BA is more effective cytokinin on shoot multiplication, KIN is more effective on shoot lenght. In spite of the increased number of shoots on media containing cytokinin, the shoot length is decreased. A negative correlation between the shoot number and their length has been observed. This kind of negative correlation was reported in Centaurea paui by using inflorescence stalk as explant [8] and C. zeybekii by axillary shoot proliferation [13].
Figure 2.
Axillary shoot proliferation on MS medium added 0.5 mgl-1BA.
Figure 3.
Cytokinin effects on axillary shoot multiplication of R.mykalea.
Figure 4.
Average shoot lengths of axillary shoots dependent on cytokinin type and concentration.
After three subculturing, solitary shoots excised from multiple shoot cultures were transferred to MS and ½ MS media containing IAA, IBA and NAA at various concentrations for rooting. Rhizogenezis was not occured MS and ½ MS medium without plant growth regulators. Auxin is necessary for in vitro rooting of R.mykalea axillary shoots. Generally, ½ MS medium added auxin is more effective than MS medium added auxin for rooting. The maximum rooting rate was obtained with half-strength MS medium supplemented with 0.5 mgl-1 IBA (55%) (Figure 5 and 6).There are many of reports about IBA is more effective than other auxins on rooting for Asteraceae such as Anthemis xylopoda [11, 23] , Centaurea spachii [12], Centaurea ragusina [24], Centaurea zeybekii [25] and Saussurea obvallata [26].
Figure 5.
Rooting of R.mykalea axillary shoots.
Figure 6.
Rooting plantlets on ½ MS medium added 0.5 mgl-1 IBA.
There was a statistically significant difference between MS and ½ MS medium on rooting. ½ MS medium is more effective than MS medium on rooting in all experiments. Also, there was a statistically significant difference on rooting of R. mykalea between auxin type and concentration.
In this study, we described a successful and rapid propagation techniques to regenerate critically endangered R.mykalea the first time by in vitro tissue culture techniques. Mature zygotic embryos isolated from achenes were used as starting material. The shoots that were obtained from in vitro germinated mature embryos were separated from primary roots and used for axillary shoot propagation. The highest axillary shoot number per explant was obtained on MS medium supplemented with 0.5 mgl-1 BA (5.8 shoot/explant). MS medium supplemented with 0.1 mgl-1 KIN was determined as the most suitable medium for the maximum shoot length (7.35 cm). Solitary shoots, removed from stock cultures, were transferred onto half-strength MS (½ MS) or MS media supplemented with various concentrations of auxins. The maximum rooting rate was obtained with half-strength MS medium supplemented with 0.5 mgl-1 IBA (55%). Rooted plantlets were transferred to external environment step by step.
The plantlets with well devoloped root were transferred to ex vitro conditions (Figure 7). Percentage of survival of shoots was approximately 60%. The appearance and growth of these plantlet were also normal.
Figure 7.
Acclimatized plantlets.
4. Conclusions
In conclusion, the present work presents a simple and successful procedure for the in vitro propagation of Rhaponticoides mykalea (Hub.-Mor.) M. V. Agab. & Greuter, a critically endangered endemic plant species.
To date there is no report on micropropagation of R. mykalea. This study is the first report on micropropagation of this species using seedlings from in vitro germinated embryos and aims to contrubute ongoing ex situ conservation programs. Additionally, this outlined protocol can be utilized as an aid in the local conservation programs to preserve this species, and it will lead for further studies on conservation and propagation of this rare and critically endangered endemic plant.
Acknowledgments
The authors are thankful to University of Adnan Menderes for financial support (Project no: FEF-07012)
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Introduction",level:"1"},{id:"sec_2",title:"2. Material and methods",level:"1"},{id:"sec_2_2",title:"2.1. Plant material and explant source",level:"2"},{id:"sec_3_2",title:"2.2. Achene viability ",level:"2"},{id:"sec_4_2",title:"2.3. Seed sterilisation, media preparation and culture conditions ",level:"2"},{id:"sec_5_2",title:"2.4. Axillary shoot proliferation",level:"2"},{id:"sec_6_2",title:"2.5. Shoot rooting and acclimatization of plantlets",level:"2"},{id:"sec_7_2",title:"2.6. Statistical analyses",level:"2"},{id:"sec_9",title:"3. Results and discussion",level:"1"},{id:"sec_10",title:"4. Conclusions",level:"1"},{id:"sec_11",title:"Acknowledgments",level:"1"},{id:"sec_11",title:"Acknowledgments",level:"2"}],chapterReferences:[{id:"B1",body:'EkimTKoyuncuMVuralMDumanHAytaçZAdigüzelNRed Data Book of Turkish Plants (Pteridophyta and Spermatophyta). Ankara-Turkey: Turkish Association for the Conservation of Nature; 2000'},{id:"B2",body:'DavisP. HFlora of Turkey and East Aegean Islands. Edinburg, U.K.: University Press; 1975'},{id:"B3",body:'HellwigF. HCentaureinae (Asteraceae) in the Mediterranean-history of ecogeographical radiation. Plant Systematics and Evolution 2004246137'},{id:"B4",body:'EmekYErdagBObservations on Kuşadası Population of Rhaponticoides mykalea, Resarch Journal of Biology Sciences 201032169174'},{id:"B5",body:'FayM. FConservation of Rare and Endangered Plants Using In Vitro Methods. In Vitro Cellular Development Biology 1992281'},{id:"B6",body:'KrogstrupPBaldurssonSNorgaardJ. VEx Situ Genetic Conservation by Use of Tissue Culture. Opera Botany 199211349\n\t\t\t'},{id:"B7",body:'FayM. FIn What Situation Is In Vitro Culture Appropriate to Plant Conservation? Biodiversity Conservation 19943176\n\t\t\t'},{id:"B8",body:'CuencaSMarcoJ. BParraRMicropropagation From Inflorescense Stems of the Spanish Endemic Plant Centaurea paui Loscos ex Wilk. (Compositae). Plants Cell Reports 199918674'},{id:"B9",body:'MurchS. JKrishnaRaj S, Saxena PK. Phytomaceuticals: Mass Production, Standardization and Conservation. Scientific Review of Alternative Medicine 2000439'},{id:"B10",body:'PhillipsG. CHubstenbergerJ. FMicropropagation by Proliferation of Axillary Buds. In: Gamborg OL., Phillips GC. (ed) Plant Cell Tissue and Organ Culture. Fundemental Methods. Germany: Springer-Verlag Berlin-Heidelberg; 19954654\n\t\t\t'},{id:"B11",body:'ErdagBEmekYIn Vitro Micropropagation of Anthemis xylopoda O.Schwarz, a Critically Endangered Species from Turkey. Pakistan Journal of Biologicial Sciences 200585691695'},{id:"B12",body:'CuencaSMarcoJ. BIn Vitro Propagation of Centaurea spachii From Inflorescence Stems. Plant Growth Regulation 20003099'},{id:"B13",body:'KurtSErdagBIn Vitro Germination and Axillary Shoot Propagation of Centaurea zeybekii. Biologia 200964197101'},{id:"B14",body:'HammattNEvansP. KThe In Vitro Propagation of an Endangered Species: Centaurea junoniana Svent. (Compositae). The Journal of Horticultural Science and Biotechnology 19856093'},{id:"B15",body:'HasançebiSTurgut Kara N, Çakır Ö,Arı Ş. Micropropagation and Root Culture of Turkish Endemic Astragalus chrysochlorus (Leguminosae). Turkish Journal of Botany 201135203'},{id:"B16",body:'IriondoJ. MPerezCMicropropagation and In Vitro Storage of Centaurium rigualii Esteve (Gentianaceae). Israel Journal of Plant Sciences 199644115'},{id:"B17",body:'Sha Valli Khan PSPrakash E, Rao KR. In Vitro Micropropagation of an Endemic Fruit Tree Syzygium alternifolium (Wight) walp. Plant Cell Reports 199716325'},{id:"B19",body:'EmekYErdagBResearchs on In Vitro Seed Germination of The Critically Endangered Endemic Plant Rhaponticoides mykalea (Hub.-Mor.) Journal of Nevsehir University of Science and Technology Institute 2012246'},{id:"B20",body:'MurashigeTSkoogFA Revised Medium For Rapid Growth And Bioassays With Tobacco Tissue Cultures. Physiologia Plantarum 196215473'},{id:"B21",body:'MiloševicMVujakovicMKaragicDVigour Tests as Indıcators of Seed Viability. Genetika 2010421103118'},{id:"B22",body:'FowlerJCohenLPractical Statistics for Field Biology. Buckingham: Open University Press; 1990'},{id:"B23",body:'RuffoniBMassaboFTissue Culture in Gerbera jamesonii hybrida. Acta Horticulturae 1991289147\n\t\t\t'},{id:"B24",body:'ErdagBEmekYAdventitious Shoot Regeneration and In Vitro Flowering of Anthemis xylopoda O. Schwarz, a Critically Endangered Turkish Endemic.Turkish Journal of Biology 2009334319326'},{id:"B25",body:'PevalekK. BIn vitro germination of Centaurea ragusina L., a Croatian Endemic Species. Acta Biologica Cracoviensia Series Botanica 19984021\n\t\t\t'},{id:"B26",body:'KurtSResearchs on In Vitro Seed Germination of The Centaurea zeybekii Wagenitz. PhD thesis. Adnan Menderes University Turkey; 2005'},{id:"B27",body:'DharUJoshiMEfficient Plant Regeneration Protocol Through Callus for Saussurea obvallata (DC.) Edgew. (Asteraceae): Effect of Explant Type, Age and Plant Growth Regulators. Plant Cell Reports 200524195'}],footnotes:[],contributors:[{corresp:"yes",contributorFullName:"Yelda Emek",address:"yelda@adu.edu.tr",affiliation:'
Department of Biology, Faculty of Arts & Science, Adnan Menderes University, Aydın, Turkey
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Wilson and Stephan Beck",authors:[{id:"176611",title:"Prof.",name:"Stephan",middleName:null,surname:"Beck",fullName:"Stephan Beck",slug:"stephan-beck"},{id:"176612",title:"Dr.",name:"Gareth A.",middleName:null,surname:"Wilson",fullName:"Gareth A. Wilson",slug:"gareth-a.-wilson"}]},{id:"49336",title:"Analysis of Next-generation Sequencing Data in Virology - Opportunities and Challenges",slug:"analysis-of-next-generation-sequencing-data-in-virology-opportunities-and-challenges",signatures:"Sunitha M. Kasibhatla, Vaishali P. Waman, Mohan M. Kale and\nUrmila Kulkarni-Kale",authors:[{id:"37914",title:"Dr.",name:"Urmila",middleName:"Dilip",surname:"Kulkarni-Kale",fullName:"Urmila Kulkarni-Kale",slug:"urmila-kulkarni-kale"}]},{id:"49316",title:"RNA-seq – Revealing Biological Insights in Bacteria",slug:"rna-seq-revealing-biological-insights-in-bacteria",signatures:"Mariana P. Santana, Flavia F. Aburjaile, Mariana T.D. Parise,\nSandeep Tiwari, Artur Silva, Vasco Azevedo and Anne Cybele Pinto",authors:[{id:"31422",title:"Prof.",name:"Vasco",middleName:null,surname:"Azevedo",fullName:"Vasco Azevedo",slug:"vasco-azevedo"},{id:"126846",title:"Dr.",name:"Anne",middleName:null,surname:"Cybelle Pinto",fullName:"Anne Cybelle Pinto",slug:"anne-cybelle-pinto"},{id:"177477",title:"MSc.",name:"Mariana",middleName:null,surname:"Santana",fullName:"Mariana Santana",slug:"mariana-santana"},{id:"177478",title:"BSc.",name:"Flavia",middleName:null,surname:"Aburjaile",fullName:"Flavia Aburjaile",slug:"flavia-aburjaile"},{id:"177479",title:"BSc.",name:"Mariana",middleName:null,surname:"Parise",fullName:"Mariana Parise",slug:"mariana-parise"},{id:"177481",title:"MSc.",name:"Sandeep",middleName:null,surname:"Tiware",fullName:"Sandeep Tiware",slug:"sandeep-tiware"},{id:"177482",title:"Prof.",name:"Artur",middleName:null,surname:"Silva",fullName:"Artur Silva",slug:"artur-silva"}]},{id:"49764",title:"Dealing with the Data Deluge – New Strategies in Prokaryotic Genome Analysis",slug:"dealing-with-the-data-deluge-new-strategies-in-prokaryotic-genome-analysis",signatures:"Leonid Zaslavsky, Stacy Ciufo, Boris Fedorov, Boris Kiryutin, Igor\nTolstoy and Tatiana Tatusova",authors:[{id:"159223",title:"Dr",name:"Tatiana",middleName:null,surname:"Tatusova",fullName:"Tatiana Tatusova",slug:"tatiana-tatusova"},{id:"177882",title:"Dr.",name:"Leonid",middleName:null,surname:"Zaslavsky",fullName:"Leonid Zaslavsky",slug:"leonid-zaslavsky"},{id:"177883",title:"Dr.",name:"Stacy",middleName:null,surname:"Ciufo",fullName:"Stacy Ciufo",slug:"stacy-ciufo"},{id:"177884",title:"Mr.",name:"Igor",middleName:null,surname:"Tolstoy",fullName:"Igor Tolstoy",slug:"igor-tolstoy"},{id:"177885",title:"Dr.",name:"Boris",middleName:null,surname:"Kiryutin",fullName:"Boris Kiryutin",slug:"boris-kiryutin"},{id:"177886",title:"Dr.",name:"Boris",middleName:null,surname:"Fedorov",fullName:"Boris Fedorov",slug:"boris-fedorov"}]},{id:"49339",title:"Reaping the Benefits of Next-generation Sequencing Technologies for Crop Improvement — Solanaceae",slug:"reaping-the-benefits-of-next-generation-sequencing-technologies-for-crop-improvement-solanaceae",signatures:"Sushil Satish Chhapekar, Rashmi Gaur, Ajay Kumar and Nirala\nRamchiary",authors:[{id:"176464",title:"Dr.",name:"Nirala",middleName:null,surname:"Ramchiary",fullName:"Nirala Ramchiary",slug:"nirala-ramchiary"},{id:"176466",title:"Dr.",name:"Rashmi",middleName:null,surname:"Gaur",fullName:"Rashmi Gaur",slug:"rashmi-gaur"},{id:"176467",title:"Mr.",name:"Ajay",middleName:null,surname:"Kumar",fullName:"Ajay Kumar",slug:"ajay-kumar"},{id:"177582",title:"Mr.",name:"Sushil Satish",middleName:null,surname:"Chhapekar",fullName:"Sushil Satish Chhapekar",slug:"sushil-satish-chhapekar"}]},{id:"49276",title:"Perspectives on the Application of Next-generation Sequencing to the Improvement of Africa’s Staple Food Crops",slug:"perspectives-on-the-application-of-next-generation-sequencing-to-the-improvement-of-africa-s-staple-",signatures:"Melaku Gedil, Morag Ferguson, Gezahegn Girma, Andreas Gisel,\nLivia Stavolone and Ismail Rabbi",authors:[{id:"96161",title:"Dr.",name:"Morag",middleName:null,surname:"Ferguson",fullName:"Morag Ferguson",slug:"morag-ferguson"},{id:"176405",title:"Dr.",name:"Melaku",middleName:null,surname:"Gedil",fullName:"Melaku Gedil",slug:"melaku-gedil"},{id:"176880",title:"Dr.",name:"Andreas",middleName:null,surname:"Gisel",fullName:"Andreas Gisel",slug:"andreas-gisel"},{id:"176881",title:"Dr.",name:"Livia",middleName:null,surname:"Stavolone",fullName:"Livia Stavolone",slug:"livia-stavolone"},{id:"176882",title:"Dr.",name:"Ismail",middleName:null,surname:"Rabbi",fullName:"Ismail Rabbi",slug:"ismail-rabbi"},{id:"176883",title:"Dr.",name:"Gezahegn",middleName:null,surname:"Girma",fullName:"Gezahegn Girma",slug:"gezahegn-girma"}]},{id:"49258",title:"Hop-variety Identification Using First- and Second-generation Sequencing",slug:"hop-variety-identification-using-first-and-second-generation-sequencing",signatures:"Hiromasa Yamauchi",authors:[{id:"176332",title:"Dr.",name:"Hiromasa",middleName:null,surname:"Yamauchi",fullName:"Hiromasa Yamauchi",slug:"hiromasa-yamauchi"}]},{id:"49529",title:"Analysis of Haplotype Sequences",slug:"analysis-of-haplotype-sequences",signatures:"Sally S. Lloyd, Edward J. Steele and Roger L. Dawkins",authors:[{id:"176478",title:"Prof.",name:"Roger",middleName:null,surname:"Dawkins",fullName:"Roger Dawkins",slug:"roger-dawkins"},{id:"177337",title:"Dr.",name:"Ted",middleName:null,surname:"Steele",fullName:"Ted Steele",slug:"ted-steele"},{id:"177338",title:"Dr.",name:"Sally",middleName:null,surname:"Lloyd",fullName:"Sally Lloyd",slug:"sally-lloyd"}]},{id:"49317",title:"On Genotyping Polymorphic HLA Genes — Ambiguities and Quality Measures Using NGS",slug:"on-genotyping-polymorphic-hla-genes-ambiguities-and-quality-measures-using-ngs",signatures:"Szilveszter Juhos, Krisztina Rigó and György Horváth",authors:[{id:"176182",title:"Dr.",name:"Szilveszter",middleName:null,surname:"Juhos",fullName:"Szilveszter Juhos",slug:"szilveszter-juhos"},{id:"176295",title:"Mr.",name:"György",middleName:null,surname:"Horváth",fullName:"György Horváth",slug:"gyorgy-horvath"},{id:"176296",title:"Ms.",name:"Krisztina",middleName:null,surname:"Rigó",fullName:"Krisztina Rigó",slug:"krisztina-rigo"}]},{id:"49528",title:"DNA-based Diagnosis of Uncharacterized Inherited Macrothrombocytopenias Using Next-generation Sequencing Technology with a Candidate Gene Array",slug:"dna-based-diagnosis-of-uncharacterized-inherited-macrothrombocytopenias-using-next-generation-sequen",signatures:"David J. Rabbolini, Marie-Christine Morel Kopp, Sara Gabrielli,\nQiang Chen, William S. Stevenson and Christopher M. Ward",authors:[{id:"169448",title:"Dr.",name:"Marie-Christine",middleName:null,surname:"Morel-Kopp",fullName:"Marie-Christine Morel-Kopp",slug:"marie-christine-morel-kopp"},{id:"169449",title:"Dr.",name:"Christopher",middleName:null,surname:"Ward",fullName:"Christopher Ward",slug:"christopher-ward"},{id:"176393",title:"Dr.",name:"David",middleName:null,surname:"Rabbolini",fullName:"David Rabbolini",slug:"david-rabbolini"},{id:"176449",title:"Ms.",name:"Sara",middleName:null,surname:"Gabrielli",fullName:"Sara Gabrielli",slug:"sara-gabrielli"},{id:"176450",title:"Mr.",name:"Qiang",middleName:null,surname:"Chen",fullName:"Qiang Chen",slug:"qiang-chen"},{id:"176451",title:"Dr.",name:"William",middleName:null,surname:"Stevenson",fullName:"William Stevenson",slug:"william-stevenson"}]},{id:"49460",title:"Clinical Implementation of Next-generation Sequencing in the Field of Prenatal Diagnostics",slug:"clinical-implementation-of-next-generation-sequencing-in-the-field-of-prenatal-diagnostics",signatures:"Gwendolin Manegold-Brauer and Olav Lapaire",authors:[{id:"176142",title:"Dr.",name:"Manegold-Brauer",middleName:null,surname:"Gwendolin",fullName:"Manegold-Brauer Gwendolin",slug:"manegold-brauer-gwendolin"},{id:"176143",title:"Dr.",name:"Olav",middleName:null,surname:"Lapaire",fullName:"Olav Lapaire",slug:"olav-lapaire"}]},{id:"48955",title:"Impact of Gene Annotation on RNA-seq Data Analysis",slug:"impact-of-gene-annotation-on-rna-seq-data-analysis",signatures:"Shanrong Zhao and Baohong Zhang",authors:[{id:"176364",title:"Dr.",name:"Shanrong",middleName:null,surname:"Zhao",fullName:"Shanrong Zhao",slug:"shanrong-zhao"}]}]}]},onlineFirst:{chapter:{type:"chapter",id:"64334",title:"Growing and Handling of Bacterial Cultures within a Shared Core Facility for Integrated Structural Biology Program",doi:"10.5772/intechopen.81932",slug:"growing-and-handling-of-bacterial-cultures-within-a-shared-core-facility-for-integrated-structural-b",body:'\n
1. Introduction
\n
Shared research core facilities can provide support to campus-wide investigators by providing research infrastructure for the production and purification of recombinant proteins for a variety of research applications. We have designed a research support structure for investigators pursuing research in structural and functional studies that require high yields of pure proteins, particularly suited for structural studies including biomolecular nuclear magnetic resonance (NMR) and small angle X-ray scattering.
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The Escherichia coli (E. coli) expression platform is commonly used for recombinant expression of proteins. The E. coli system has several advantages over yeast, insect cells, or mammalian cell expression systems: E. coli are relatively easy to handle, the doubling time is short, media are low-cost and there are abundantly established methods for protein expression [1, 2, 3, 4]. The E. coli expression platform is also well-suited for stable isotope labeling of proteins for biological NMR studies [5, 6, 7, 8, 9]. Structural studies of proteins demand large quantities of high purity protein. Meeting these requirements can be challenging, however, advancements in high-throughput technologies for recombinant expression of proteins have greatly advanced in the last decade or more, in large part due to efforts from large structural genomics and structural proteomics centers [1, 4, 10, 11, 12]. The lessons learned and technologies developed from these centers can allow for rapid assessment of different expression strategies, which can be transferred and scaled down to smaller-scale centers and academic labs [3, 4, 13].
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In addition to a demand for large quantities of highly pure protein, structural studies also often demand high solubility and stability of the protein in solution. To address this need, a high-throughput fluorescence-based thermal-shift assay, also known as differential scanning fluorimetry (DSF), has been implemented at the large structural genomics and structural proteomics centers [14]. DSF was originally developed as a high-throughput drug discovery assay to screen for small molecules that bind to and stabilize target proteins [15, 16, 17]. The DSF screen has been further adapted to optimize buffer conditions by varying the pH, buffer components, detergents, reducing agents and small molecules to screen for conditions that increase the stability and conformational homogeneity of a protein [14, 17, 18, 19, 20], which is key in obtaining high-quality structural data.
\n
We have established and optimized standard operating procedures for growing and handling bacterial cultures in a shared core laboratory to support Integrative Structural Biology and have used these in our own research [21, 22, 23, 24, 25, 26, 27, 28, 29]. The Integrative Structural Biology effort within the Biomolecular Research Center, a shared core facility, allows researchers at Boise State University and collaborating institutions to generate new knowledge about protein and RNA structure and function. We aim to understand how biomolecules assemble into stable structures and how structural dynamics can impact their function. Here we describe specific procedures for growing and handling bacterial cultures for overexpression and isolation of recombinant proteins, 15N/13C uniform labeling of recombinant proteins, protein isolation and purification, and analysis of protein solubility that are ideal for implementation in a shared research core laboratory that serves a multitude of diverse customers and research laboratories. Here we outline a general workflow of essential steps in protein expression and purification that includes plasmid amplification, mini-expression screening, optimized larger-scale protein production, protein isolation and purification, and characterization of optimized experimental solution buffer conditions (Figure 1).
\n
Figure 1.
A protein expression and purification workflow from plasmid to stable purified protein.
\n
\n
2. Materials
\n
All reagents listed in this chapter are commonly available from commercial vendors. A chemical hygiene plan including storage, shelf life, and safety of all chemicals should be in place at the institution.
\n
2.1 Preparation of chemically competent cells
\n
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Ice.
Lysogeny broth (LB) medium: 10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl. Sterilize by autoclaving and store at room temperature.
Super optimal broth (SOB, a.k.a. Hanahan’s Broth) medium for DH5α cells: 20 g/L tryptone, 5 g/L yeast extract, 0.5 g/L NaCl, 0.186 g/L KCl. Adjust the pH to 7.0 with NaOH. Sterilize by autoclaving and store at room temperature.
Culture tubes and flasks.
Incubator/shaker.
Centrifuge tubes.
Serological pipettes.
Repeating pipettor.
Dimethyl sulfoxide (DMSO).
Competent Cell (CC) buffer: 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 15 mM CaCl2, 55 mM MnCl2, 250 mM KCl, pH 6.7. Dissolve all components except MnCl2 and adjust the pH to 6.7 with KOH. Then add the MnCl2 and filter sterilize the solution over a 0.22 μm filter.
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\n
2.2 Transformation of cells for expression of desired plasmid
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\n
Ice.
LB or super optimal broth with catabolite repression (SOC).
Culture tubes and flasks.
Incubator/shaker.
Centrifuge tubes.
Serological pipettes.
Competent cells.
LB-agar plates containing the appropriate antibiotic.
Plasmid DNA.
Heat block set.
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\n
2.3 Calculating efficiency of competent cells
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\n
Transformed colonies on LB-agar plate (see Section 3.3).
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\n
2.4 Inoculating overnight cultures
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LB.
15 mL conical tube.
Sterile inoculating loop.
Appropriate antibiotics.
Shaker/incubator.
Sterile aluminum foil or culture tube cap.
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2.5 Glycerol stocks
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50% glycerol solution (autoclaved).
Make the 50% glycerol solution by diluting 100% glycerol into water.
Screwtop cryogenic vials.
Liquid nitrogen.
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\n
2.6 DNA plasmid purification
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Resuspension buffer: 50 mM Tris-HCl, pH 8.0; 10 mM ethylenediaminetetraacetic acid (EDTA), 20 μg/mL RNase A.
Precipitation buffer: 3 M potassium acetate, 2 M glacial acetic acid, 4°C.
Wash buffer: 70% ethanol.
95% (or 100%) ethanol.
TE buffer: 10 mM Tris-HCl, pH 8.0; 0.1 mM EDTA.
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\n
2.7 Testing for protein expression and solubility in E. coli
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\n
LB.
Appropriate antibiotics.
Incubator/shaker.
Microcentrifuge tubes.
Centrifuge.
Isopropyl β-D-1-thiogalactopyranoside (IPTG).
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\n
2.8 Lysing cells
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Induced cells suspended in lysis buffer with a protease inhibitor cocktail, 0.1 mg/mL DNase I, 1 mg/mL lysozyme and 0.1 mg/mL 4-(2-Aminoethyl) benzenesulfonyl fluoride (AEBSF).
Sonication buffer.
Ice-saltwater bath.
Probe sonicator equipped with ½ inch tip.
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2.9 Gel electrophoresis, protein quantification
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Electrophoresis system.
4–12% Bis-Tris mini gel.
Sample loading buffer: 10% glycerol, 0.14 M Tris Base, 0.1 M Tris-HCl, 2% lithium dodecyl sulfate (LDS), 0.5 mM EDTA, 0.02% Blue G250; 0.006% phenol red, 1.25% 2-mercaptoethanol, pH 8.5.
Running buffer: 50 mM 2-(N-morpholino)ethanesulfonic acid (MES), 50 mM Tris base, 0.1% SDS, 1 mM EDTA, pH 7.2.
Coomassie Blue stain.
Protein molecular weight marker.
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\n
2.10 Testing lysis conditions for solubility
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Buffer A: 50 mM Tris pH 7.5, 100 mM NaCl, 5 mM EDTA, 1 mg/mL lysozyme.
Buffer B: 50 mM Tris pH 7.5, 2 M NaCl, 5 mM EDTA, 1 mg/mL lysozyme.
Buffer C: 50 mM Tris pH 7.5, 100 mM NaCl, 50% detergent, 1 mg/mL lysozyme.
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\n
2.11 Large-scale expression of recombinant proteins
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LB.
IPTG.
Culture tubes and flasks.
Incubator/shaker.
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2.12 Uniform 15N/13C labeling of recombinant proteins
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LB.
IPTG.
Culture tubes and flasks.
Incubator/shaker.
10X M9 medium: 340 mM Na2HPO4, 220 mM KH2PO4, 85.5 mM NaCl, pH 7.4.
2.13 Protein purification using immobilized metal affinity chromotography (IMAC)
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Immobilized metal affinity chromatography (IMAC) is a common method for affinity purification. A genetically encoded 6-histidine repeat affinity tag can be introduced to the carboxy or amino terminal end of the protein during cloning, which has high affinity for metal ions. The protocol given here is for affinity purification by immobilization of nickel ions with a chelator molecule, nitrilotriacetic acid (NTA) that is covalently bound to agarose; commonly known as Ni-NTA agarose. The following buffers are meant to represent a general starting point. Depending on the pI of your recombinant protein and the propensity to nonspecifically interact with the column material or resident E. coli proteins, modifications may need to be made. Additional purification may be necessary, especially when purifying proteins that bind to nucleic acids. A lithium wash may be added to the Ni-NTA purification to remove nucleic acids. Ion exchange, heparin affinity, size exclusion chromatography are often added in addition to a nickel affinity purification step.
Lysis buffer: 0.1 M Tris-HCl, 0.1 M NaCl, pH 8.1.
Wash buffer: Lysis buffer plus 5–20 mM imidazole.
Elution buffer: Lysis buffer plus 100–300 mM imidazole.
Probe sonicator.
1 mg/mL lysozyme.
Protease inhibitor cocktail.
AEBSF.
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\n
2.14 Differential scanning fluorimetry to assess protein stability
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Low ionic strength buffer (e.g., 10 mM Tris-HCl).
qPCR machine with filter set that matches fluorescent dye and equipped with a ramp rate of minimum 1°C/min.
Inoculate 5 mL of LB (or SOB if preparing DH5α cells) with 10 μL of appropriate E. coli cells2 and grow overnight at 37°C and 250 rpm in a shaking incubator.
Use the overnight culture to inoculate 250 mL of LB (or SOB if preparing DH5α cells) and incubate at 30°C until the optical density at 600 nm (OD600) is between 0.4–0.6.
Chill the culture for at least 10 min on ice. For steps 4–10, keep the cell suspension on ice.
Spin the cell suspension for 10 min at 6000× g.
Gently resuspend the pellet in 50 mL ice-cold CC buffer into 50-mL conical tubes. Resuspend with a 10-mL serological pipette and avoid introducing bubbles.
Incubate the cell suspension on ice for at least 10 min.
Spin for 10 min at 6000× g at 4°C.
Gently resuspend the pellet in 9.4 mL ice-cold CC buffer and add 0.7 mL DMSO.
Incubate the cell suspension on ice for at least 10 min.
Distribute the cell suspension in 50–200 μL aliquots in 1.5-mL microcentrifuge tubes.3
Flash freeze the cell suspension in liquid nitrogen and store the tubes at −80°C.
At −80°C the cells will be competent for at least 6 months.
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\n
3.2 Transformation of E. coli cells with plasmid DNA
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\n
Take competent cells out of −80°C and thaw on ice (approximately 20–30 min).
For each transformation, remove two LB-agar plates (containing the appropriate antibiotic) from storage at 4°C and warm to room temperature; optionally warm to 37°C in an incubator.
Mix 10–100 pg. DNA into 20–50 μL of competent cells in a 1.5 mL microcentrifuge tube.
Gently mix by flicking the bottom of the tube with your finger a few times.
Incubate the competent cell/DNA mixture on ice for 20–30 min.
Heat shock each tube at 42°C for 45–60 s.
Put the tubes back on ice for 2 min.
Add 1 mL of LB medium (without antibiotic) to the bacteria and grow at 37°C and 250 rpm in a shaking incubator for 45 min.
Plate 50 μL of the transformed cells onto one of the 10 cm LB-agar plate containing the appropriate antibiotic and the remaining 950 μL onto the second 10 cm LB-agar plate.
Incubate plates at 37°C overnight.
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\n
3.3 Calculating transformation efficiency of competent cells
\n
\n
Count the number of colony forming units (CFUs) on the LB-agar plate after transformation (see Section 3.2).
Calculate the transformation efficiency (TrEff) in CFUs/μg of DNA using Eq. (1).
Add 5–10 mL of liquid LB to a culture tube and add the appropriate antibiotic to at correct concentration. A good negative control is LB media plus antibiotic without any bacteria inoculated. You should see no growth in this culture after overnight incubation.
Using a sterile inoculating loop, select a single colony from your LB-agar plate for plasmid purifications and a swipe from 10 to 20 colonies for protein expression (Section 3.2).
Add the inoculating loop to the liquid LB with antibiotics and swirl.
Loosely cover the culture with sterile aluminum foil or a culture tube cap.
After incubation, check for growth, which is characterized by a cloudy haze in the media.
For overnight cultures, incubate bacterial culture at 30°C for 12–16 h in a shaking incubator.4
For long-term storage of the bacteria, you can proceed with Section 3.5.
\n
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3.5 Preparation of a glycerol stock
\n
\n
Follow Section 3.2 for transforming and plating E. coli cells.
Follow Section 3.4 for inoculating an overnight culture.
Add 500 μL of the overnight culture to 500 μL of 50% glycerol in a 2 mL screw top cryogenic vial5 and gently mix.
Submerse the glycerol stock tube into liquid nitrogen and store at −80°C. The stock is now stable for years, as long as it is kept at −80°C. Subsequent freeze and thaw cycles reduce shelf life.
To recover bacteria from your glycerol stock: open the tube and use a sterile loop, toothpick, or pipette tip to scrape some of the frozen bacteria off of the top. Do not let the glycerol stock thaw. Streak the bacteria onto an LB-agar plate.
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3.6 Plasmid DNA purification
\n
\n
Preheat the TE Buffer in the incubator at 37°C.
Spin 5 mL of the overnight LB culture at 6000× g for 10 min at 4°C. Discard supernatant.
Add 250 μL Resuspension Buffer containing RNase A to the cell pellet and resuspend the pellet by pipetting until homogeneous.
Add 250 μL Lysis Buffer. Mix gently by inverting the capped tube until homogeneous. Do not vortex. Incubate the tube at room temperature for 5 min.
Add 350 μL Precipitation Buffer. Mix immediately by inverting the tube until homogeneous. Do not vortex. Centrifuge the lysate at 16,000× g for 10 min at 4°C.
Add 2–2.5 volumes 95% or 100% ethanol and 1/10 volume of 3 M Na-acetate (pH 4.8) to the supernatant. Invert the microcentrifuge tube to mix. Let stand for 2 min at room temperature.
Centrifuge solution at high speed (at least 16,000× g) for 15–30 min at 4°C. Discard supernatant.
Open and invert the tube on a paper towel to drain it out.
Wash pellet by adding 500 μL cold 70% ethanol.
Centrifuge solution at high speed (at least 16,000× g) for 5 min at room temp. Discard supernatant by pipetting it out of the tube.
Dry the pellet by inverting over paper towel for 5–20 min.
Resuspend dry DNA with TE.
Store plasmid DNA at 4°C (short term) or store the DNA in aliquots at −20°C (long term.)
\n
\n
3.7 Testing for soluble protein expression in E. coli
\n
The following protocol is written for proteins expressed under the control of the lac, tac, or T7 promoters. The method as described is a generic protocol that can be expanded to test expression in different strains of E. coli, induction temperatures, concentrations of IPTG, or even in the presence of ligands or cofactors.
\n
3.7.1 Protein expression
\n
\n
Transform plasmid into an E. coli expression strain following Section 3.2.
Inoculate a liquid LB culture following Section 3.4.
Grow cells for a few hours at 37°C, shaking at 250 rpm. Make sure the tubes are tilted.
Monitor the turbidity. Once the culture reaches an OD600 of 0.4–0.6 (takes ~2–4 h, depending on the sample), take out 2 mL of the culture. Measure the actual OD600.
Transfer the equivalent of 1 mL of cells at OD600 = 0.8 in a 1.5-mL microcentrifuge tube.6
Collect cells by centrifugation at 16,000× g on a tabletop centrifuge for at least 1 min. Carefully remove all of the supernatant. This is the uninduced sample. Store the cells at −20°C.
Add IPTG to a final concentration of 1.0 mM to the remaining culture. Continue shaking at 250 rpm for 12–16 h at 18°C.
Measure the OD600. Collect cells by centrifugation in two tubes containing the equivalent of 1 mL of cells at OD600 = 0.8 and remove the supernatant. These are the induced samples; one tube will be used to test for expression and the second for solubility. Store the cells at −20°C until ready to test for expression.
\n
\n
3.7.2 Testing for expression
\n
\n
Take the tube of uninduced and one tube of induced cells and resuspend each in 100 μL of 1X SDS polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer.
Boil the samples for 10 min, then cool down to room temperature.
Centrifuge for 5 min at 16,000× g on a tabletop centrifuge at room temperature.
Take 10 μL of each sample from the top of tube taking care not to disturb the pellet.
Analyze the results using SDS-PAGE following Section 3.9 (Figure 2), with western blotting if necessary.
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Figure 2.
SDS-PAGE gel of pre- and post-induction of an RNA binding protein (RBP) in both rich (LB) and minimal (M9) media. The arrow indicates the recombinant RBP.
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\n
3.7.3 Testing for solubility
\n
\n
Resuspend the remaining induced cell pellet in 50 μL of lysis buffer containing protease inhibitors and 1 mg/mL of lysozyme.
Follow Section 3.8.1 for freeze-thawing to lyse the cells.
Spin down in a microcentrifuge at maximum speed for 10 min at 4°C.
Carefully transfer all of the supernatant into a new microcentrifuge tube. Add 50 μL of 2X SDS-PAGE buffer. This is the soluble fraction.
Resuspend the pellet in 100 μL of 1X SDS-PAGE buffer. This is the insoluble fraction.
Boil the samples for 10 min, then cool down to room temperature.
Centrifuge for 5 min at 16,000× g at room temperature.
Analyze 15 μL of each sample using SDS-PAGE following Section 3.9.
\n
\n
\n
3.8 Lysing cells
\n
Traditionally cell lysis can be done with physical disruption or reagent-based methods. Freeze-thaw protocol works best for small volumes (less than 1 mL) in 1.5 mL microcentrifuge tubes. Sonication can be done with smaller volumes using a microtip.
\n
3.8.1 Freeze-thaw
\n
\n
Freeze the samples to be lysed (typically 0.1–1.0 mL in a 1.5 mL microcentrifuge tube) in a − 80°C freezer, leave for 15 min.
Thaw immediately in a 42°C water bath. Vortex vigorously to mix well.
Repeat the two previous steps three more times (four freeze-thaw-vortex cycles in all).
Spin the tubes for 5 min at maximum speed in a microcentrifuge.
Separate the supernatant (contains soluble protein) from the pellet (contains insoluble protein) by pipetting out the supernatant to a clean tube.
\n
\n
3.8.2 Sonication
\n
\n
Prepare ice-saltwater bath by sprinkling salt over packed ice in a container.
Place a 50-mL conical tube containing the cell pellet suspended in lysis buffer securely in the ice-saltwater bath.
Insert clean tip of a sonicator in the sample without contacting sides or bottom of the tube.
Set the output power, cycle, and timer to the optimal settings (e.g., five short bursts of 15 s followed by intervals of 30 s for cooling).
Keep the suspension at all times on ice.
\n
\n
\n
3.9 Gel electrophoresis
\n
\n
Add ~100 μg of protein to SDS sample buffer.
Heat the sample at 70°C for 10 min.
Load the entire volume of sample onto a 4–12% Bis-Tris mini gel.
Run the gel at 200 V for 35 min.
At the end of the electrophoresis, wash the gel in deionized water three times.
Stain the gel with Coomassie Blue stain for 1 h.
Wash the gel with deionized water extensively until the water is clear.
\n
\n
3.10 Testing lysis conditions for solubility
\n
The solubility of a protein depends strongly on the composition of the lysis buffer. Using the procedure described below, the solubility of a specific protein can be tested under neutral (Buffer A), high salt (Buffer B), and with detergent included (Buffer C).
Follow Section 3.7.1 for the best expressing condition and collect four induced samples.
Spin down the cells for 5 min at 12,000× g in a microcentrifuge.
To each cell pellet, add 100 μL of the appropriate buffer (see Section 2.10).
Vortex to resuspend the cells.
Lyse cells using the freeze-thaw method (Section 3.8.1).
To the supernatant, add 25 μL of 4X SDS-PAGE loading buffer.
To the cell pellet, add 125 μL of 1X SDS-PAGE loading buffer.
Heat all samples to 95°C for 5 min.
Vortex briefly and then centrifuge for 5 min at maximum speed.
Load 20 μL on an SDS-PAGE gel; avoid disturbing the pellet.
\n
\n
3.11 Large-scale expression of recombinant proteins
\n
\n
Transform plasmid into an E. coli expression strain following Section 3.2.
Inoculate a liquid LB culture for an overnight growth following Section 3.4.
The next day, use the overnight culture to inoculate 1 L of LB with the appropriate antibiotic.
Grow cultures at 37°C and 250 rpm shaking until the OD600 is ~0.6–0.8.
Induce expression of protein by adding IPTG to a final concentration of 0.1 mM.
Lower the temperature to 18°C and continue 250 rpm shaking for 12–16 h.
Follow Sections 3.7.1 and 3.7.2 to test for protein expression.
Harvest the cells by centrifugation at 6000× g.
Suspend cells in lysis buffer and store at −20°C.
\n
\n
3.12 Uniform 15N/13C labeling of recombinant proteins
\n
This protocol is for proteins expressed under the control of the lac, tac, or T7 promoters.
\n
3.12.1 Day 1
\n
\n
Transform 10 μL of competent BL21(DE3) cells (or derivatives) with 10 ng of plasmid DNA and plate cells on LB-agar containing the appropriate antibiotics (See Section 3.2).
\n
\n
3.12.2 Day 2
\n
\n
Prepare 50 mL of unlabeled defined medium for overnight culture as follows, in a 200 mL culture flask:\n
5 mL 10X M9 medium.
5 mL 10X ammonium chloride.
0.75 mL 20% glucose.
50 μL of each CaCl2, MgSO4, thiamine and biotin.
antibiotic at working concentration.
autoclaved water to 50 mL.
Inoculate a 5 mL culture (LB with appropriate antibiotic) with several freshly grown colonies (ca. 10–20).
Incubate cells in tilted tubes for a few hours at 37°C and 250 rpm in a shaking incubator, until the culture is visibly turbid.
Prewarm 50 mL of unlabeled defined medium to 30°C. While warming, centrifuge the LB culture (5 min, 4000× g, 30°C) and discard supernatant.
Resuspend cell pellet in 50 mL unlabeled defined media, for a starting OD600 of ~0.03–0.08. Grow the culture overnight at 30°C in a shaking incubator.
\n
\n
3.12.3 Day 3
\n
\n
Prepare 500 mL of 13C, 15N labeled defined medium as follows, in a 2 L baffled flask:\n
50 mL 10X M9 medium.
0.5 g 15NH4Cl dissolved in 5 mL water and sterile filtered.
1.5 g 13C glucose dissolved in 10 mL water and sterile filtered.
500 μL of each CaCl2, MgSO4, thiamine and biotin.
antibiotic at working concentration.
autoclaved water to 500 mL.
Prewarm the 500 mL of 13C, 15N labeled defined medium.
Centrifuge the overnight 50-mL unlabeled defined medium culture (5 min, 4000× g, 30°C) and discard supernatant.
Resuspend the cell pellet in 500 mL of 13C, 15N labeled defined medium, for a starting OD600 of 0.03–0.08.
Grow culture at 37°C and 250 rpm in a shaking incubator until cells reach mid-log growth (OD600 ~ 0.5–0.8).
Once cells reach mid-log growth (OD600 ~ 0.5–0.8), measure the OD600. Calculate the corrected volume (in mL) to take for the sample aliquot equivalent of 1 mL of cells at OD600 = 0.8 (See Section 3.7.1 for details).
Transfer aliquot to a microcentrifuge tube, and spin it down at maximum speed for at least 1 min at room temperature. Remove the supernatant. This is an uninduced sample. Store the uninduced cells at −20°C.
Induce protein expression by adding IPTG based on the optimal values of IPTG concentration, incubation time and incubation temperature (See Section 3.7).
After the induced cells have grown for the proper length of time, dilute 200 μL of the culture 10-fold with 1X PBS and measure the OD600. To prepare an induced sample, take an aliquot containing the equivalent of 1 mL of cells at OD600 = 0.8 and immediately process it as described in Section 3.7.2.
Harvest the cells by centrifugation at 6000× g for 20–30 min at 4°C. Discard supernatant. Store the pellet at −20°C until ready for cell lysis.
\n
\n
\n
3.13 Protein purification using IMAC
\n
\n
Resuspend cell pellet in ~35 mL of lysis buffer containing AEBSF, a protease inhibitor cocktail, and 1 mg/mL lysozyme.
Lyse cells (See Section 3.8).
Remove 75 μL of lysate and pipette into a 1.5 mL microcentrifuge tube. Centrifuge the 75 μL aliquot for 10 min at 12,000× g at room temperature.
Separate the supernatant into a new vial and treat with 25 μL of 4X SDS PAGE sample buffer. To the remaining pellet, add 100 μL of 1X SDS PAGE.
Boil separated and SDS buffer-treated samples for 10 min and store at room temperature for further SDS-PAGE analysis.
Centrifuge the remaining lysate (ca. 35 mL) at 16,000× g at 4°C for 20–30 min.
Filter the supernatant over a 0.4-micron syringe filter.
Pre-equilibrate the appropriate amount of Ni-NTA agarose for the amount of protein expressed in desired equilibration buffer; typically, 1–2 mL of settled agarose washed with two column volumes (CVs) of sterile, deionized water followed by two CVs of the buffer.
Bind the filtered lysate to the Ni-NTA agarose either batch or column loading. For batch loading, combine the filtered lysate and Ni-NTA agarose and gently rock for 30–60 min prior to pouring into the column. For column loading, pack Ni-NTA agarose into the column and pass the filtered lysate through the column. Collect the flow through eluent for SDS-PAGE analysis.
Wash the column with 15 CVs of cold lysis buffer. Collect wash eluent for SDS-PAGE analysis.
A step gradient consisting of 15 CVs each of 5, 10, and 20 mM imidazole may be used to determine best washing conditions. Collect wash eluents for SDS-PAGE analysis.
Wash the column with 20 CVs elution buffer, collecting 1 mL fractions.
Evaluate all collected samples by SDS-PAGE (see Section 3.9)7 (Figure 3).
Pool fractions containing pure recombinant protein and dialyze into an appropriate buffer.
Clean Ni-NTA agarose by washing with 0.5 M NaOH for 30 min. Wash with five CVs sterile deionized water and store in 30% ethanol for long-term storage. The Ni-NTA agarose may be re-used for the same protein multiple times.
\n
Figure 3.
SDS-PAGE gel of a typical Ni-NTA purification of an RBP (arrow indicates the recombinant RBP). (A) Samples appear in the following order: MW markers, Lysate, Supernatant, Pellet, Flowthrough, Wash #1: 50 mM Tris-Cl, 100 mM NaCl, pH 7.7; washes #2–4: 10 mM Imidazole, 50 mM Tris-Cl, 100 mM NaCl, pH 7.7. (B) MW markers, Elutions #1–9: 200 mM Imidazole, 50 mM Tris-Cl, 100 mM NaCl, pH 7.7. Some protein elutes from the column in the wash steps. All fractions are kept and can be pooled after SDS-PAGE analysis.
\n
\n
3.14 Differential scanning fluorimetry to assess protein stability
\n
\n
Prepare 1500 μL of 18 μM protein in dilution buffer.
Add 1.5 μL of 5000X dye.
Pipette up and down gently to mix.
Divide the protein plus dye solution among 10 vials: 140 μL per vial (some stock solution will remain).
Add 80 μL additive to be screened to one of nine vials.
Add 80 μL of dilution buffer in the tenth vial as a control.
Pipette up and down gently to mix.
Transfer 50 μL of protein plus dye plus additive solution (or control solution) to the 96-well PCR plate in triplicate.8
Cover PCR plate with a sheet of optically clear adhesive and seal each well.
Spin 96-well PCR plate in a centrifuge equipped with a plate holder at 800× g for 2 min at room temperature.
Place 96-well PCR plate into qPCR machine and run the following program:\n
select total volume per well 50 μL
select experiment type melting curve
set the following temperatures: an initial 2 min hold at 25°C, increase in increments of 0.5–1.0°C and hold each for 30 s,9 to a final temperature of 95°C (with a 2 min hold).
Export data for further analysis.
Data can be plotted as the fluorescence vs. temperature (Figure 4).
After buffer optimization, structural data can be collected such as a 1H, 15N 2D NMR spectrum (see Figure 5 for example of spectrum).
\n
Figure 4.
DSF analysis of an RBP in buffer (10 mM Tris-Cl) with different additives. (A) A graph of the fluorescence at 602 nm at increasing temperatures for the surveyed additive screen. The inflection point preceding the peak is the melting temperature. (B) A first derivative plot with a four-point smoothing applied helps to visualize the melting temperature, where the peak is the melting temperature. The legend provides a key for both A and B.
\n
Figure 5.
1H, 15N 2D NMR spectrum of an RBP prepared using the methods described here.
\n
\n
\n
4. Conclusion
\n
We have described the workflow for protein expression and purification used in our shared core laboratory. These methods for growing and handling bacterial cultures work well for plasmid amplification, mini-expression screening, optimized larger-scale protein production, protein isolation and purification, and characterization of optimized experimental solution buffer conditions. Future methods can be added as needed by the users of the core and the university research community.
\n
Acknowledgments
\n
This publication was made possible by Institutional Development Awards (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grants P20GM109095 and P20GM103408. The authors wish to acknowledge Jackson Wall for careful reading and suggestions.
\n
Conflict of interest
Authors have no conflict of interest.
\n',keywords:"protein expression, recombinant protein, isotype enrichment, core facility, protocols",chapterPDFUrl:"https://cdn.intechopen.com/pdfs/64334.pdf",chapterXML:"https://mts.intechopen.com/source/xml/64334.xml",downloadPdfUrl:"/chapter/pdf-download/64334",previewPdfUrl:"/chapter/pdf-preview/64334",totalDownloads:196,totalViews:0,totalCrossrefCites:0,dateSubmitted:"February 19th 2018",dateReviewed:"October 10th 2018",datePrePublished:"November 8th 2018",datePublished:null,readingETA:"0",abstract:"We have established and optimized standard operating procedures for growing and handling bacterial cultures in a shared core laboratory to support Integrative Structural Biology. The Integrative Structural Biology effort within the Biomolecular Research Center allows researchers to generate new knowledge about protein and RNA structure and function. We aim to understand how biomolecules assemble into stable structures and how structural dynamics impacts their function. Here we describe specific procedures for growing and handling bacterial cultures for overexpression and isolation of recombinant proteins, 15N/13C uniform labeling of recombinant proteins, protein isolation and purification, and analysis of protein solubility that are ideal for implementation in a shared research core laboratory that serves a multitude of diverse customers and research laboratories.",reviewType:"peer-reviewed",bibtexUrl:"/chapter/bibtex/64334",risUrl:"/chapter/ris/64334",signatures:"Lisa R. Warner, Olga Mass, Nancy Donnelly Lenn, Briana R. Grantham and Julia Thom Oxford",book:{id:"7240",title:"Growing and Handling of Bacterial Cultures",subtitle:null,fullTitle:"Growing and Handling of Bacterial Cultures",slug:"growing-and-handling-of-bacterial-cultures",publishedDate:"December 4th 2019",bookSignature:"Madhusmita Mishra",coverURL:"https://cdn.intechopen.com/books/images_new/7240.jpg",licenceType:"CC BY 3.0",editedByType:"Edited by",editors:[{id:"204267",title:"Dr.",name:"Madhusmita",middleName:null,surname:"Mishra",slug:"madhusmita-mishra",fullName:"Madhusmita Mishra"}],productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"}},authors:null,sections:[{id:"sec_1",title:"1. Introduction",level:"1"},{id:"sec_2",title:"2. Materials",level:"1"},{id:"sec_2_2",title:"2.1 Preparation of chemically competent cells",level:"2"},{id:"sec_3_2",title:"2.2 Transformation of cells for expression of desired plasmid",level:"2"},{id:"sec_4_2",title:"2.3 Calculating efficiency of competent cells",level:"2"},{id:"sec_5_2",title:"2.4 Inoculating overnight cultures",level:"2"},{id:"sec_6_2",title:"2.5 Glycerol stocks",level:"2"},{id:"sec_7_2",title:"2.6 DNA plasmid purification",level:"2"},{id:"sec_8_2",title:"2.7 Testing for protein expression and solubility in E. coli",level:"2"},{id:"sec_9_2",title:"2.8 Lysing cells",level:"2"},{id:"sec_10_2",title:"2.9 Gel electrophoresis, protein quantification",level:"2"},{id:"sec_11_2",title:"2.10 Testing lysis conditions for solubility",level:"2"},{id:"sec_12_2",title:"2.11 Large-scale expression of recombinant proteins",level:"2"},{id:"sec_13_2",title:"2.12 Uniform 15N/13C labeling of recombinant proteins",level:"2"},{id:"sec_14_2",title:"2.13 Protein purification using immobilized metal affinity chromotography (IMAC)",level:"2"},{id:"sec_15_2",title:"2.14 Differential scanning fluorimetry to assess protein stability",level:"2"},{id:"sec_17",title:"3. Methods",level:"1"},{id:"sec_17_2",title:"3.1 Preparation of chemically competent cells",level:"2"},{id:"sec_18_2",title:"3.2 Transformation of E. coli cells with plasmid DNA",level:"2"},{id:"sec_19_2",title:"3.3 Calculating transformation efficiency of competent cells",level:"2"},{id:"sec_20_2",title:"3.4 Inoculating cultures",level:"2"},{id:"sec_21_2",title:"3.5 Preparation of a glycerol stock",level:"2"},{id:"sec_22_2",title:"3.6 Plasmid DNA purification",level:"2"},{id:"sec_23_2",title:"3.7 Testing for soluble protein expression in E. coli",level:"2"},{id:"sec_23_3",title:"3.7.1 Protein expression",level:"3"},{id:"sec_24_3",title:"3.7.2 Testing for expression",level:"3"},{id:"sec_25_3",title:"3.7.3 Testing for solubility",level:"3"},{id:"sec_27_2",title:"3.8 Lysing cells",level:"2"},{id:"sec_27_3",title:"3.8.1 Freeze-thaw",level:"3"},{id:"sec_28_3",title:"3.8.2 Sonication",level:"3"},{id:"sec_30_2",title:"3.9 Gel electrophoresis",level:"2"},{id:"sec_31_2",title:"3.10 Testing lysis conditions for solubility",level:"2"},{id:"sec_32_2",title:"3.11 Large-scale expression of recombinant proteins",level:"2"},{id:"sec_33_2",title:"3.12 Uniform 15N/13C labeling of recombinant proteins",level:"2"},{id:"sec_33_3",title:"3.12.1 Day 1",level:"3"},{id:"sec_34_3",title:"3.12.2 Day 2",level:"3"},{id:"sec_35_3",title:"3.12.3 Day 3",level:"3"},{id:"sec_37_2",title:"3.13 Protein purification using IMAC",level:"2"},{id:"sec_38_2",title:"3.14 Differential scanning fluorimetry to assess protein stability",level:"2"},{id:"sec_40",title:"4. Conclusion",level:"1"},{id:"sec_41",title:"Acknowledgments",level:"1"},{id:"sec_44",title:"Conflict of interest",level:"1"}],chapterReferences:[{id:"B1",body:'Vincentelli R, Romier C. Expression in Escherichia coli: Becoming faster and more complex. Current Opinion in Structural Biology. 2013;23(3):326-334'},{id:"B2",body:'Rosano GL, Ceccarelli EA. Recombinant protein expression in Escherichia coli: Advances and challenges. Frontiers in Microbiology. 2014;5:172'},{id:"B3",body:'Vincentelli R, Bignon C, Gruez A, Canaan S, Sulzenbacher G, Tegoni M, et al. Medium-Scale Structural Genomics: Strategies for Protein Expression and Crystallization.Accounts of Chemical Research. 2003;36(3):165-172'},{id:"B4",body:'Berrow NS, Büssow K, Coutard B, Diprose J, Ekberg M, Folkers GE, et al. Recombinant protein expression and solubility screening in Escherichia coli: A comparative study. Acta Crystallogr Sect D Biol Crystallogr. 2006;62(10):1218-1226'},{id:"B5",body:'Muchmore DC, McIntosh LP, Russell CB, Anderson DE, Dahlquist FW. Expression and nitrogen-15 labeling of proteins for proton and nitrogen-15 nuclear magnetic resonance. Methods in Enzymology. 1989;177:44-73'},{id:"B6",body:'Fiaux J, Bertelsen EB, Horwich AL, Wüthrich K. Uniform and residue-specific 15N-labeling of proteins on a highly deuterated background. Journal of Biomolecular NMR. 2004;29(3):289-297'},{id:"B7",body:'Skrisovska L, Schubert M, Allain FH-T. Recent advances in segmental isotope labeling of proteins: NMR applications to large proteins and glycoproteins. Journal of Biomolecular NMR. 2010;46(1):51-65'},{id:"B8",body:'Tugarinov V, Kay LE. Ile, Leu, and Val methyl assignments of the 723-residue malate synthase G using a new labeling strategy and novel NMR methods. Journal of the American Chemical Society. 2003;125(45):13868-13878'},{id:"B9",body:'Freiburger L, Sonntag M, Hennig J, Li J, Zou P, Sattler M. Efficient segmental isotope labeling of multi-domain proteins using Sortase A. Journal of Biomolecular NMR. 2015;63(1):1-8'},{id:"B10",body:'Edwards AM, Arrowsmith CH, Christendat D, Dharamsi A, Friesen JD, Greenblatt JF, et al. Protein production: Feeding the crystallographers and NMR spectroscopists. Nature Structural Biology. 2000;7:970-972'},{id:"B11",body:'Yee A, Gutmanas A, Arrowsmith CH. Solution NMR in structural genomics. Current Opinion in Structural Biology. 2006;16(5):611-617'},{id:"B12",body:'Busso D, Thierry JC, Moras D. The Structural Biology and Genomics Platform in Strasbourg: An Overview. Methods in Molecular Biology. New York, NY: Humana Press; 2008. pp. 523-536'},{id:"B13",body:'Gräslund S, Nordlund P, Weigelt J, Hallberg BM, Bray J, Gileadi O, et al. Protein production and purification. Nature Methods. 2008;5(2):135-146'},{id:"B14",body:'Reinhard L, Mayerhofer H, Geerlof A, Mueller-Dieckmann J, Weiss MS. Optimization of protein buffer cocktails using Thermofluor. Acta Crystallographica. Section F, Structural Biology and Crystallization Communications. 2013;69(Pt 2):209-214'},{id:"B15",body:'Niesen FH, Berglund H, Vedadi M. The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nature Protocols. 2007;2(9):2212-2221'},{id:"B16",body:'Pantoliano MW, Petrella EC, Kwasnoski JD, Lobanov VS, Myslik J, Graf E, et al. High-density miniaturized thermal shift assays as a general strategy for drug discovery. Journal of Biomolecular Screening. 2001;6(6):429-440'},{id:"B17",body:'Senisterra GA, Markin E, Yamazaki K, Hui R, Vedadi M, Awrey DE. Screening for ligands using a generic and high-throughput light-scattering-based assay. Journal of Biomolecular Screening. 2006;11(8):940-948'},{id:"B18",body:'Nettleship JE, Brown J, Groves MR, Geerlof A. Methods for Protein Characterization by Mass Spectrometry, Thermal Shift (ThermoFluor) Assay, and Multiangle or Static Light Scattering. New York, NY: Humana Press; 2008. pp. 299-318'},{id:"B19",body:'Ericsson UB, Hallberg BM, DeTitta GT, Dekker N, Nordlund P. Thermofluor-based high-throughput stability optimization of proteins for structural studies. Analytical Biochemistry. 2006;357(2):289-298'},{id:"B20",body:'Vedadi M, Niesen FH, Allali-Hassani A, Fedorov OY, Finerty PJ, Wasney GA, et al. Chemical screening methods to identify ligands that promote protein stability, protein crystallization, and structure determination. Proceedings of the National Academy of Sciences of the United States of America. 2006;103(43):15835-15840'},{id:"B21",body:'Fallahi A, Kroll B, Warner LR, Oxford RJ, Irwin KM, Mercer LM, et al. Structural model of the amino propeptide of collagen XI alpha1 chain with similarity to the LNS domains. Protein Science. 2005;14(6):1526-1537'},{id:"B22",body:'Warner LR, Fallahi A, Kroll B, Irwin KM, Yingst S, Mercer LM, et al. Modeling and characterization of the amino propeptide of collagen α1(XI), a regulatory domain in collagen fibrillar architecture. Materials Research Society Symposium Proceedings, Structure and Mechanical Behavior of Biological Materials. 2005;874:41-46'},{id:"B23",body:'Oxford JT, DeScala J, Morris N, Gregory K, Medeck R, Irwin K, et al. Interaction between amino propeptides of type XI procollagen alpha1 chains. The Journal of Biological Chemistry. 2004;279(12):10939-10945'},{id:"B24",body:'Medeck RJ, Sosa S, Morris N, Oxford JT. BMP-1-mediated proteolytic processing of alternatively spliced isoforms of collagen type XI. The Biochemical Journal. 2003;376(pt 2):361-368'},{id:"B25",body:'Warner LR, Brown RJ, Yingst SM, Oxford JT. Isoform-specific heparan sulfate binding within the amino-terminal noncollagenous domain of collagen α1(XI). The Journal of Biological Chemistry. 2006;281(51):39507-39516'},{id:"B26",body:'Ryan RE, Martin B, Mellor L, Jacob RB, Tawara K, McDougal OM, et al. Oncostatin M binds to extracellular matrix in a bioactive conformation: Implications for inflammation and metastasis. Cytokine. 2015;72(1):71-85'},{id:"B27",body:'Kahler RA, Yingst SMC, Hoeppner LH, Jensen ED, Krawczak D, Oxford JT, et al. Collagen 11a1 is indirectly activated by lymphocyte enhancer-binding factor 1 (Lef1) and negatively regulates osteoblast maturation. Matrix Biology. 2008;27(4):330-338'},{id:"B28",body:'Oxford JT, DeScala J, Morris N, Gregory K, Medeck R, Irwin K, et al. Interaction between amino propeptides of type XI procollagen α1 chains. The Journal of Biological Chemistry. 2004;279(12):10939-10945'},{id:"B29",body:'Gregory KE, Oxford JT, Chen Y, Gambee JE, Gygi SP, Aebersold R, et al. Structural organization of distinct domains within the non-collagenous N-terminal region of collagen type XI. The Journal of Biological Chemistry. 2000;275(15):11498-11506'}],footnotes:[{id:"fn1",explanation:"The stock solution of 10 mg/mL is above the solubility limit of biotin, do not sterile filter this solution. Simply make the solution with previously sterilized water."},{id:"fn2",explanation:"Some cell lines have a resident plasmid, such as BL21(DE3) pLysS or pLysE cells and require addition of antibiotics for selection of cells containing those plasmids."},{id:"fn3",explanation:"A repeating pipettor or a multichannel pipettor speeds up the aliquoting process greatly. This will minimize the time that the competent cells are manipulated, thus increasing their competency. Expect competency of ca. 107–108 CFU/μg of plasmid DNA."},{id:"fn4",explanation:"For some applications (especially culturing cells in minimal defined media) cultures should never be overgrown; growing overnight cultures at a reduced temperature, 25–30°C, is suggested."},{id:"fn5",explanation:"Snap top tubes are not recommended."},{id:"fn6",explanation:"In order to have equal loading on an SDS-PAGE gel, the same amount of cells need to be harvested for gel analysis. To harvest the same number of cells each time, calculate the volume in mL needed of your culture that would be the equivalent of 1 mL of OD600 = 0.8. E.g. X mL = 0.8/OD600 of your culture."},{id:"fn7",explanation:"Store lysate cell pellet at −20°C until SDS-PAGE has confirmed that full extraction of desired protein from the pellet is accomplished. Keep all buffers and protein samples at 4°C during purification. Batch vs. column choice will depend on binding properties of the individual protein. The SDS-PAGE of the step gradient imidazole washes will illustrate what is the highest imidazole concentration that can be used as an initial wash to clean off non-binding contaminants. If large amounts of protein remain in the cell pellet, alternative growing methods, such as IPTG concentration adjustment, or alternative purification methods including purification under denaturing conditions, should be considered. Additional purification may be necessary, such as ion exchange, or heparin binding column chromatography."},{id:"fn8",explanation:"Excess solutions are suggested to account for loss due to transfers and sticking to the sidewall of the tubes."},{id:"fn9",explanation:"Slower ramp rates will provide better melting temperature resolution, however not all instruments are equipped with fine temperature resolution."}],contributors:[{corresp:null,contributorFullName:"Lisa R. Warner",address:null,affiliation:'
Biomolecular Research Center, Boise State University, Boise, Idaho, USA
Biomolecular Research Center, Boise State University, Boise, Idaho, USA
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