Autoinduction Media Preparation: Media should be prepared with the sterile solutions and sterile equipment obtained in Week 1.
ADVICE: All of these steps should be done using sterile technique with a flame on. Both the pipettes and the benchtop should be cleaned with water and ethanol before starting. Make sure to only use sterile tips, flasks, pipettes, water, and media.
Preparation of Autoinduction Media for Expressions (100 mL) Scale to make amount you need for your experiment based on the following amounts per 100 mL. Prepare fresh and do not plan on storing extra AIM.
Aspartate (5%, pH 7.5) 5 mL
Glycerol (10%) 5 mL
25x Mineral Salts 4 mL
Glucose (40%) 0.125 mL
MgSO4 (1 M) 0.2 mL
Arabinose (20%) 0.25 mL
Trace metals (5000x) 20 microliter
18 AA mix (25x) stored @ 4°C 4 mL
Add appropriate antibiotics (ampicillin to final concentration of 100 microgram/mL and tetracycline to 25 microgram/mL).
Add sterile water to a final volume of 100 mL.
Recommended volumes and flasks for the expressions during weeks 2 and 3:
Wild-type protein 75 mL expression in a 250 mL flask
ncAA protein 1 75 mL expression in a 250 mL flask
ncAA protein 1 (negative control -ncAA) 5 mL expression in culture tube
ncAA protein 2 75 mL expression in a 250 mL flask
ncAA protein 2 (negative control -ncAA) 5 mL expression in culture tube
sfGFP wild-type control (-ncAA) 5 mL expression in culture tube
ADVICE (EXAMPLE FOR 250 mL—SCALE ACCORDING TO YOUR NEEDS): It is convenient to start with a 250 mL bottle of sterile water and remove about 50 mL of it by placing it into another sterile vessel (for later use). Then all of the media components can be added to the 200 mL of sterile water and topped off with sterile water to a final volume of 250 mL. Then add 250 microliter of 100 mg/mL ampicillin to the media, and then place 75 mL of it in a 250 mL baffled flask for expression of wild-type protein. 175 microliter of 25 mg/mL tetracycline can then be added to the remaining media (175 mL) and separated into expression flasks. ncAAs will be added later.
Each lab section should use the appropriate code of tape color to label flasks so it is easy to recover them. Remember there are 4 sections, over 80 students and some weeks 80-100 flasks growing.
Addition of Cells from Starter Cultures:
To the prepared flasks (75 mL media/flask), saturated cell cultures in non-inducing media of the appropriate cell line must be added to start the expressions. Dilute cells of appropriate overnight starter culture 1:100-1:200 to each flask containing 75 mL. Incubate these cultures for 0.5 -1 hr before adding the ncAA (this incubation may be shortened for certain ncAAs).
ncAA Preparation and Addition: (Week 3)
The concentration recommended for the ncAA is >1 mM final ncAA concentration in the expression media. The ncAAs may need assistance dissolving before adding to the media, depending on the chemical characteristics of the ncAA. It is recommended to weigh out slightly more than the appropriate amount of ncAA directly into a microcentrifuge tube. Start by adding 0.5 mL of sterile water and mixing – if it dissolves, add it to the appropriate expression flask. If most of the ncAA dissolves, first try adding an additional 0.5 mL of sterile water. If very little is dissolved after the first 1 mL, try adding 1 molar equivalent of NaOH from the 8 M stock solution (add 5 µL 8 N NaOH at a time and do not exceed 20 µL total). Adding an excess of NaOH can damage some of the ncAAs and cause problems with cell growth. Once ncAAs are added to their appropriate flasks, let them incubate while shaking at 250-300 rpm at 37°C for up to 48 hours (this long incubation is designed to meet the schedule of BB 494; typically cultures are grown for 24-40 hours before harvesting).
To obtain a zero time point of protein expression, remove 250 microliter of cell culture from the original starter cells in non-inducing medium and centrifuge at 3000 – 5000 rcf for 5-10 min. Discard the supernatant and store the small cell pellet at -20°C in your freezer box (label all samples well, so you and your partners can identify in the future).
Express protein for 24-48 hours. Before harvesting cells by centrifugation, remember to remove 250 microliter cells for final time points for a crude gel first. Also, determine the OD600 of the cultures. Divide each cell sample into 25-35 mL aliquots in pre-weighed conical tubes. Spin the cells in 50 mL conical tubes for 10 min at 5000-8000 rcf. in the bench top centrifuge (not the Sorvall). Remove the supernatant by pouring it off, determine the mass of each cell pellet, and store the cell pellets at -80 °C. Again label tubes well with section, group number, date, and sample. Store cell pellets in small Ziploc bags that are well labeled with pen directly on the bag with your lab section, group number, and date. Do not use tape to label tubes or bags—it falls off at -80 °C.
Deep Thoughts on Bacterial Strains and Plasmids
We are providing you with starters grown in non-inducing media with which to inoculate your larger cultures but what exactly are you using? Are you blindly taking what the TA or instructor gives you and following the instructions for expressing protein in this manual or do you know how the system works and what exactly is in that starter culture?
If you are adding antibiotics to your media, why are you doing it? How do you know what the correct antibiotics are? Not all of your expressions use the same antibiotics and why is this? How is resistance to these antibiotics imparted?
There are a huge variety of different bacterial strains we could be using, each with unique features optimized for specific purposes and with advantages and disadvantages. Some are designed to tightly control expression and are used for expressing proteins that may be toxic to a cell. Others lack or contain mutants of certain genes in order to prevent protein degradation, promote disulfide bond formation, or limit production of certain metabolites. What bacterial strain are you using? What are some of its advantages and disadvantage? Why do you think it may have been chosen for these experiments?
Due to the time constraints of this course, the bacteria containing the appropriate plasmids were also provided for you. Just because you did not carry out this part of the experiment yourself doesn’t mean you don’t have to understand how it was done. How were these plasmids generated? What do each of the plasmids contain? How did these plasmids get into the bacteria where your protein of interest can be overexpressed?
At the very least, you’ll want to know and understand: the type of E.coli you are using, the plasmids you are using, and what is encoded on each of these plasmids.