Difference between revisions of "20.109(S08):Induce protein expression (Day4)"

Revision as of 23:15, 7 January 2008

Introduction

Remind how IPTG works; discuss places where protein folding/production may go wrong; probably discuss sequencing on this day.

Protocols

Another option: start inducing all four mutants, then analyze sequencing data while IPTG induction runs. Less pressure this way - have another exercise as a back-up...

Part 1: Cell measurement and IPTG induction

1. For each mutant protein, you will be given a 6 mL aliquot of DE3 cells carrying the mutant plasmid; you will also receive a tube of DE3 carrying wild-type inverse pericam. These cells should be in or close to the mid-log phase of growth for good induction, just as they were for transformation. Like last time, check the OD₆₀₀ value of your cells until it falls between 04.and 06. (Better to overshoot a little than undershoot.)
2. Once your cells have reached the appropriate growth phase, set aside 1.5 mL of cells from each tube as a control (no IPTG) sample. Then take an aliquot of cold IPTG (0.1 M), and add to your remaining cells at a final concentration of 1 mM. You should prepare four mutant and one wild-type tube.
3. Return your tubes to the 37 °C incubator, and note down the time.
4. While your IPTG-stimulated cells are producing protein, you will analyze the sequence data for the plasmids they are carrying. At the end of the day, you will choose only one colony from each mutant to save, and give the other sample to the teaching faculty to be bleached and thrown away.

Part 2: Analyze sequence data

Your goal today is to analyze the sequencing data for four samples - two independent colonies from each of two mutants - and then decide which two colonies to proceed with. You will want to have this document handy, and mark the expected location of your mutation with bold text before proceeding. (Just compare to your annotation of the old IPC sequence document, using Word Count or the Find tool.) The new document contains the inverse pericam sequence as before, but also the flanking sequences before and after IPC, which are separated from IPC by a blank line. The second page of the document contains the reverse complement of IPC-in-pRSET, which was gotten using this website. Be sure to bold your mutant codon in the reverse complement sequence as well.

The data from the MIT Biopolymers Facility is available at this link. Choose the "Login to dnaLIMS" link and then use "astachow" and "be109" to login. At the bottom of the left panel should be a link to download your sequencing results. Select the appropriate order # (you'll be told which one is correct) and then "submit." The quickest way to start working with your data is to follow the "view" link. From this link you'll see the sequencing traces and can click on on "sequence text" to view it.

Rather than look through the sequence to magically find the relevant portion, you can align the data you just got with the standard inverse pericam sequence and the differences will be quickly identified. There are several web-based programs for aligning sequences and still more programs that can be purchased. The steps for using one web-based tool are sketched below.

Align with "bl2seq" from NCBI

1. The alignment program can be accessed through the NCBI BLAST page or directly from this link
2. To allow for gaps in the sequence alignment, uncheck the "filter" box. All the other default settings should be fine.
3. Paste the sequence text from your sequencing run into the "Sequence 1" box. This will now be the "query." If there were ambiguous areas of your sequencing results, these will be listed as "N" rather than "A" "T" "G" or "C" and it's fine to include Ns in the query.
4. Paste the inverse pericam sequence into the "Sequence 2" box. For samples probed with the forward primer, use the regular IPC sequence; for those using a reverse primer, you should put in the reverse complement. Which alignment will be more useful depends on the location of your mutant.
5. Click on Align. Matches will be shown by vertical lines between the aligned sequences. You should see a long stream of matches, followed by lots of errors in the last ~200bp of the sequence – ignore the error-ridden part of the data, as it may not accurately reflect your mutant plasmid. In this stream of matches, the the 1-3 missing lines indicating your mutant codon should stand out. If they don’t, use the numbering or Find tool to locate the appropriate codon.
6. You should print a screenshot of each alignment to pdf (and to paper if you desire). These will be used to prepare a figure showing what you found today. You might want to email yourself the alignment screen shots or post them to your wiki userpage.

If both colonies for a given mutant have the correct sequence, flip a coin and proceed with one or the other; ditto if both are inconclusive or clearly wrong. If one appears right and the other doesn’t, of course proceed with the former.

For your reference, another popular sequence alignment program is "CLUSTAL-W" from EMBL-EBI , located here.

Part 3: MATLAB Intro?

Make available a self-guided tour to introduce students to MATLAB (used on Day 7)? With sequencing timing being a question mark, probably not good to have Atissa come on this day.

Part 4: Cell observation and collection

1. After ~2 hours, you will pour 1.5 mL from each tube into a labeled eppendorf, then spin for 1 min. at maximum speed. Save the other 3 mL!
2. Aspirate the supernatant from each eppendorf, using a fresh yellow pipet tip on the end of the glass pipet each time.
3. Observe the colour of each of your pellets. If both the wild-type and the mutant pellets appear yellow-greenish to the eye, proceed as follows:
   • Do NOT toss the rest of the liquid. First, ,easured the OD₆₀₀ according to part 5.
   • Next, pour 1.5 mL more liquid culture on top of the pellet, spin again, and aspirate the supernatant.
   • The last 1.5 mL of culture may be bleached and discarded.
4. If one or more of your pellets are white or only dimly coloured, please ask one of the teaching staff too show you the room temperature rotary shaker in the back room. You will continue to grow your bacteria overnight. Tomorrow morning, the teaching staff will collect your pellets for you and freeze them. As you can see above, the +IPTG pellets are from 3 mL of culture, while the -IPTG pellets come from 1.5 mL of culture.

Part 5: Preparation for next time

Next time, you will lyse your bacterial samples to release their proteins, and run these out on a protein gel. In order to compare the amount of protein in the -IPTG versus +IPTG samples, you would like to normalize by the number of cells. At this point, you may have only three samples ready (-IPTG only), or you may have all six. In either case, measure the OD₆₀₀ of a 1:10 dilution of cells for each finished sample, and write this number down in your notebook. Then spin down the cells and aspirate the supernatant.

For next time

Start working on Part 3 of portfolio (something like: a short piece on a fluorescence-enabled technology), encourage them to draft Part 1 from their outline as well…