Growing and Handling of Bacterial Cultures within a Shared Core Facility for Integrated Structural Biology Program

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, 15 N/ 13 C 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. pipettor speeds up the aliquoting process minimize the time that the competent cells are manipulated, their competency. competency of ca. 10 7 –10 8 CFU/ μ g of plasmid DNA.


Introduction
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.
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].
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.
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, 15 N/ 13 C 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 8. 100 mM CaCl 2. 10. 10 mg/mL thiamine. 11. 10 mg/mL biotin. 1 12. Antibiotic for plasmid selection.

Protein purification using immobilized metal affinity chromotography (IMAC)
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 1 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.
to remove nucleic acids. Ion exchange, heparin affinity, size exclusion chromatography are often added in addition to a nickel affinity purification step. 2.14 Differential scanning fluorimetry to assess protein stability 1. Low ionic strength buffer (e.g., 10 mM Tris-HCl).
2. qPCR machine with filter set that matches fluorescent dye and equipped with a ramp rate of minimum 1°C/min.

3.
A fluorescent dye that will bind proteins.

5.
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.
6. Incubate the cell suspension on ice for at least 10 min.
8. Gently resuspend the pellet in 9.4 mL ice-cold CC buffer and add 0.7 mL DMSO.
9. Incubate the cell suspension on ice for at least 10 min.
10. Distribute the cell suspension in 50-200 μL aliquots in 1.5-mL microcentrifuge tubes. 3 11. Flash freeze the cell suspension in liquid nitrogen and store the tubes at −80°C.
12. At −80°C the cells will be competent for at least 6 months.

Transformation of E. coli cells with plasmid DNA
1. Take competent cells out of −80°C and thaw on ice (approximately 20-30 min).
2. 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.
7. Put the tubes back on ice for 2 min.
8. 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.
9. 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. 10. Incubate plates at 37°C overnight.

Calculating transformation efficiency of competent cells
1. Count the number of colony forming units (CFUs) on the LB-agar plate after transformation (see Section 3.2).

Inoculating cultures
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.
2. 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).
3. Add the inoculating loop to the liquid LB with antibiotics and swirl.
4. Loosely cover the culture with sterile aluminum foil or a culture tube cap.
5. After incubation, check for growth, which is characterized by a cloudy haze in the media.
6. For overnight cultures, incubate bacterial culture at 30°C for 12-16 h in a shaking incubator. 4 7. For long-term storage of the bacteria, you can proceed with Section 3.5.

Preparation of a glycerol stock
1. Follow Section 3.2 for transforming and plating E. coli cells.
2. Follow Section 3.4 for inoculating an overnight culture.
3. Add 500 μL of the overnight culture to 500 μL of 50% glycerol in a 2 mL screw top cryogenic vial 5  11. Dry the pellet by inverting over paper towel for 5-20 min.
13. Store plasmid DNA at 4°C (short term) or store the DNA in aliquots at −20°C (long term.)

Testing for soluble protein expression in E. coli
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.

Protein expression
1. Transform plasmid into an E. coli expression strain following Section 3.2.
2. Inoculate a liquid LB culture following Section 3.4.  5. Transfer the equivalent of 1 mL of cells at OD 600 = 0.8 in a 1.5-mL microcentrifuge tube. 6 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.

Grow cells for a few hours at
7. 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.
8. Measure the OD 600 . Collect cells by centrifugation in two tubes containing the equivalent of 1 mL of cells at OD 600 = 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.

Testing for expression
1. 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.
2. Boil the samples for 10 min, then cool down to room temperature.
3. Centrifuge for 5 min at 16,000× g on a tabletop centrifuge at room temperature.
4. Take 10 μL of each sample from the top of tube taking care not to disturb the pellet.
5. Analyze the results using SDS-PAGE following Section 3.9 (Figure 2), with western blotting if necessary.

Testing for solubility
1. Resuspend the remaining induced cell pellet in 50 μL of lysis buffer containing protease inhibitors and 1 mg/mL of lysozyme.
2. Follow Section 3.8.1 for freeze-thawing to lyse the cells.
3. Spin down in a microcentrifuge at maximum speed for 10 min at 4°C. 4. Carefully transfer all of the supernatant into a new microcentrifuge tube. Add 50 μL of 2X SDS-PAGE buffer. This is the soluble fraction.
5. Resuspend the pellet in 100 μL of 1X SDS-PAGE buffer. This is the insoluble fraction.
6. Boil the samples for 10 min, then cool down to room temperature. 7. Centrifuge for 5 min at 16,000× g at room temperature.
8. Analyze 15 μL of each sample using SDS-PAGE following Section 3.9.

Lysing cells
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.

Freeze-thaw
1. 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.
2. Thaw immediately in a 42°C water bath. Vortex vigorously to mix well.
3. Repeat the two previous steps three more times (four freeze-thaw-vortex cycles in all).
4. Spin the tubes for 5 min at maximum speed in a microcentrifuge.
5. Separate the supernatant (contains soluble protein) from the pellet (contains insoluble protein) by pipetting out the supernatant to a clean tube.

Sonication
1. Prepare ice-saltwater bath by sprinkling salt over packed ice in a container.
2. Place a 50-mL conical tube containing the cell pellet suspended in lysis buffer securely in the ice-saltwater bath.  8. Harvest the cells by centrifugation at 6000× g.
9. Suspend cells in lysis buffer and store at −20°C.

Uniform 15 N/ 13 C labeling of recombinant proteins
This protocol is for proteins expressed under the control of the lac, tac, or T7 promoters.

Day 2
1. Prepare 50 mL of unlabeled defined medium for overnight culture as follows, in a 200 mL culture flask: • 5 mL 10X M9 medium.
• antibiotic at working concentration.
5. Resuspend cell pellet in 50 mL unlabeled defined media, for a starting OD 600 of ~0.03-0.08. Grow the culture overnight at 30°C in a shaking incubator.
• 0.5 g 15 NH 4 Cl dissolved in 5 mL water and sterile filtered.
• 1.5 g 13 C glucose dissolved in 10 mL water and sterile filtered.
• antibiotic at working concentration.
6. Once cells reach mid-log growth (OD 600 ~ 0.5-0.8), measure the OD 600 . Calculate the corrected volume (in mL) to take for the sample aliquot equivalent of 1 mL of cells at OD 600 = 0.8 (See Section 3.7.1 for details).
7. 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.
8. I nduce protein expression by adding IPTG based on the optimal values of IPTG concentration, incubation time and incubation temperature (See Section 3.7).
9. 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 OD 600 . To prepare an induced sample, take an aliquot containing the equivalent of 1 mL of cells at OD 600 = 0.8 and immediately process it as described in Section 3.7.2.
10. 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.

Protein purification using IMAC
1. Resuspend cell pellet in ~35 mL of lysis buffer containing AEBSF, a protease inhibitor cocktail, and 1 mg/mL lysozyme.

Conclusion
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   © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 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.