The appropriate proteins will be produced by growing cultures of E. coli DH10B cells, which have different plasmids in them depending on the ability needed. To ensure that only cells with the appropriate plasmids can grow in the media, they must be grown with antibiotics present. The wild-type protein will be produced from a pBad plasmid (pBad-gene), and cells containing pBad need to be grown in the presence of ampicillin. The ncAA-mutant protein will also be produced from a pBad plasmid, which contains the same gene but with a TAG site at one of the codons (pBad-TAG-gene). Furthermore, in order to produce proteins with non-canonical amino acid, one also needs to add unnatural translational machinery. This unnatural machinery is on the pDule-RS plasmid, and cells containing pDule need to be grown in the presence of tetracycline. In addition, the non-canonical amino acid must be supplied in the media because the E. coli cells do not produce the ncAAs naturally. Therefore, while the wild-type protein producing cells only need to be grown in the presence of ampicillin to produce wild-type protein, the ncAA-mutant protein producing cells will need to be grown with ampicillin, tetracycline, and non-canonical amino acid in the media in order to produce ncAA-mutant protein.
As a further control of monitoring protein production, negative control expressions must be run for each type of ncAA used. These expressions ensure that no natural amino acids are being used by the synthetase and inserted into the TAG site. Negative control expressions lack ncAA in the media; therefore, only truncated proteins should be produced by these expressions. As a positive control for your media, you will express 5 mL cultures of a superfolder green fluorescent (sfGFP) protein construct. These cells will contain only the pBad-sfGFP plasmid (amp only) and will turn green if the media was prepared correctly.
After approximately 24-48 hours of expression, the large volume cell cultures should be saturated with cells that have been induced to overexpress the protein of interest. Separating the cells from the media and storing the cell pellets at -80 °C after the expression is complete will allow the protein to be purified from the cells at a later time.
Note: This year we are staggering the expression and purification of wild-type and ncAA-mutant proteins to allow you to spend more time understanding your protein and developing an experimental hypothesis. Teams will just grow cultures of their wild-type protein and a sfGFP control during week 2 and then the entire set of wild-type and ncAA-mutant proteins during week 3. Scale the media components appropriately.
Remember these instructions for volumes to prepare are guidelines—make sure you understand the principles and can adjust how much media you make and the volume of cultures you grow accordingly. Do not prepare more media than you need for that week’s expression.
Equipment for 50 mL expression cultures
- 250 mL sterile baffled flasks (one for wild-type, two for ncAA-mutant proteins)
- Two sterile culture tubes for negative control expression
- Sterile bottles (150 mL, 250 mL, 500 mL)
- Sterile pipette tips
- Large centrifuge
- Sterile 1.5 mL microcentrifuge tubes
- Oakridge centrifuge tubes
- Sterile conical 50 mL centrifuge tubes
- Shaker (temperature set to 37 °C)
- Shaker clamps (3 small) for each group—TAs and instructors will provide these
- Appropriate starter cell cultures grown in non-inducing media (See Appendix 4)
- Sterile H2O
- Aspartate (5%, pH 7.5)
- Glycerol (10%)
- 18 AA mix (25 x) (stored at 4 °C)
- 25 x Mineral Salts
- Arabinose (20%) (stored at -20 °C)
- MgSO4 (1 M)
- Glucose (40%)
- Trace metals (5000x)
- Ampicillin (1000x stock) (100 mg/mL in H2O) (stored at -20 °C)
- Tetracycline (1000x stock) (25 mg/mL in DMF) (stored at -20 °C)
- 8 M NaOH
- Non-canonical amino acids of interest
Suggested Resources and Protocols
- Media preparation and protein expression: A basic outline for this procedure can be found below. For a more in-depth discussion on the production, monitoring, and purification of ncAA-mutant proteins, see the following article:
Hammill, J.T.; Miyake-Stoner, S.; Hazen, J.L.; Jackson, J.C; Mehl, R.A. (2007) Preparation of site-specifically labeled fluorinated proteins for 19F-NMR structural characterization. Nature Protocols 2(10), 2601-2607.
- Selection of polyacrylamide gels: The following resource from Bio-Rad contain relevant concepts for protein gel electrophoresis and can be found in the literature resource area on Canvas:
Deep Thoughts on Exploiting the Central Dogma
The basic details of genetic code expansion are detailed in the Background of this lab manual. The goal of genetic code expansion is to site-specifically incorporate a non-canonical amino acid into your protein of choice. How exactly does this work? How is the genetic code facilitated through tRNA? What is the anticodon on the orthogonal tRNA? Where in the protein is your non-canonical amino acid incorporated?
Genetic code expansion requires at least four different components. What are these components? Where do you need them to be? How are these components made? What would happen if any of these components were missing? For example, what would happen if the non-canonical amino acid were not included in your media? Or if the orthogonal tRNA synthetase were missing or an incompatible orthogonal tRNA synthetase included?
Thinking about these questions can also help you troubleshoot your expression if things do not seem to be going according to plan.
Bio-Rad Mini Protean Tetra Cell handbook (in particular Section 4.2 contains protocols for making stock solutions for discontinuous Laemmli SDS-PAGE gels and buffers).
Deep Thoughts on Sterile Technique
Maintaining sterile technique is a key part of working with E. coli and molecular biology in general. You want your desired E. coli to grow and nothing else. But germs are everywhere! And what wouldn’t want to grow in a nice, warm, aerated nutrient rich broth? In order to prevent other things from growing, we use sterile technique (ie. killing anything we don’t want with heat or ethanol). Practicing good sterile technique will first and foremost help you in the laboratory setting.
But beyond this lab course, sterile technique may prove to be useful to you. Consider any sort of medical practice- whether you are pre-medicine, pre-dental, or someone who will likely visit a doctor at some point in their life- sterile technique limits the spread of pathogens and reduces your chances of developing a harmful disease. Consider carefully how to organize your workspace and tools to maintain sterility.