20.109(S16):Induce protein expression (Day5)

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20.109(S16): Laboratory Fundamentals of Biological Engineering

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Introduction

IPTG.png
Lactose(lac).png

Last time you transformed your mutant DNA into BL21(DE3)pLysS cells. The colonies that arose were used to inoculate liquid cultures, grown to saturation overnight, and then sub-cultured. Today you will add IPTG to these sub-cultures in mid-log phase to induce protein expression. Next time you will purify the resultant protein. We won’t shy away from telling you that there are many things that can go wrong at this stage! However, each one is certainly a learning experience.

As evidenced by Nagai’s work, wild-type inverse pericam is not toxic to BL21(DE3)pLysS cells. Although it is unlikely that your point mutation will dramatically change this fact, in general a novel protein may turn out to be toxic. If this is the case, only very small amounts of protein are produced before the bacteria die. Keep in mind that overexpressing a single protein may come at the expense of producing proteins needed for survival, and will most likely cause cell death eventually; however, toxic proteins hasten this demise. Aberrant toxicity can sometimes be alleviated by reducing the culture temperature (e.g., to 30 °C).

Based on its fluorescence activity, wild-type inverse pericam allows proper folding of (cp)EYFP, and based on its response to calcium, it also allows calmodulin to fold. One problem you may encounter is that your mutant proteins will no longer fold correctly. Since you made mutations in the calcium sensor part of IPC, rather than the fluorescent part, it is unlikely that your protein will destroy EYFP fluorescence. However, a common problem with misfolded proteins is the formation of insoluble aggregates, due for instance to improperly exposed hydrophobic surfaces. Proteins can be purified from these aggregates – called inclusion bodies – but the process is more labor-intensive than for soluble proteins. (The proteins must be extracted under more harsh conditions than you will use next time, then purified under denaturing conditions, before finally attempting to renature the proteins.) Inclusion bodies sometimes form simply due to very high expression of the protein of interest, causing it to pass its solubility limit. This outcome can be prevented by lowering the culture temperature, the induction duration, the amount of IPTG, or the growth phase of the bacteria.

One final point to keep in mind is that not all proteins can be produced in bacteria. Eukaryotic proteins that require post-translational modifications (such as glycosylation) for activity require eukaryotic hosts (such as yeast, or the commonly used CHO – Chinese hamster ovary – cells). Sometimes eukaryote-derived proteins will be truncated or otherwise mistranslated by E. coli due to differential codon bias; errors in translation can be prevented by providing additional tRNAs to the culture or directly to the bacteria via plasmids. Despite all this complexity, prokaryotic hosts have been plenty good enough to produce proteins for certain therapies, notably the cytokine G-CSF. This cytokine is taken by patients needing to replenish their white blood cells (e.g., after chemotherapy), and sold as Neupogen by the company Amgen.

Sequence trace data
Normal bases versus chain-terminating bases
Sequencing gel


After you induce your cells with IPTG, you will let the protein factories do their work for 2-3 hours. During this time, you will evaluate the DNA from your two X#Z candidates. The invention of automated sequencing machines has made sequence determination a relatively fast and inexpensive endeavor. The method for sequencing DNA is not new but automation of the process is recent, developed in conjunction with the massive genome sequencing efforts of the 1990s. At the heart of sequencing reactions is chemistry worked out by Fred Sanger in the 1970s which uses dideoxynucleotides (see schematic above left). These chain-terminating bases can be added to a growing chain of DNA but cannot be further extended. Performing four reactions, each with a different chain-terminating base, generates fragments of different lengths ending at G, A, T, or C. The fragments, once separated by size, reflect the DNA’s sequence. In the “old days” (all of 15-20 years ago!) radioactive material was incorporated into the elongating DNA fragments so they could be visualized on X-ray film (image above center). More recently fluorescent dyes, one color linked to each dideoxy-base, have been used instead. The four colored fragments can be passed through capillaries to a computer that can read the output and trace the color intensities detected (image above right). Your sample was sequenced in this way by Genewiz on an ABI 3730x1 DNA Analyzer.

Analysis of sequence data is no small task. “Sequence gazing” can swallow hours of time with little or no results. There are also many web-based programs to decipher patterns. The nucleotide or its translated protein can be examined in this way. Thanks to the genome sequence information that is now available, a new verb, “to BLAST,” has been coined to describe the comparison of your own sequence to sequences from other organisms. BLAST is an acronym for Basic Local Alignment Search Tool, and can be accessed through the National Center for Biotechnology Information (NCBI) home page.

Today you will carefully examine the results of your sequencing reactions and determine which of your two samples contains the wanted mutation (and no unwanted random mutations). Next time you will purify your mutated protein.

Protocols

Part 1: Cell measurement and IPTG induction

  1. Obtain your 6 mL aliquot of BL21(DE3)pLysS cells carrying each mutant plasmid (X#Z 1 and 2) and an aliquot with 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 values of your cells (650-700 μL of a 1:10 dilution) until they fall between 0.4 and 0.8.
    • OD values at the higher end should favor more protein production.
  2. Once your cells have reached the appropriate growth phase, set aside - on ice - 1.5 mL of cells from each tube as a no-induction control (no IPTG) sample. You can pellet these cells now or later in the class when you pellet your IPTG-stimulated cells.
  3. Take an aliquot of cold IPTG (0.1 M), and add to your remaining 4.5 mL of cells at a final concentration of 1 mM. You should prepare two mutant and one wild-type tube.
  4. Return your tubes to the rotary shaker in the 37 °C incubator, and note down the time.
  5. While your IPTG-stimulated cells are producing protein, you will analyze the sequence data and restriction digests of the plasmids they are carrying. At the end of the day, you will choose only one of your X#Z candidates to save (the one that contains your mutation), and aspirate the other into your bleach flask.

Part 2: Analyze sequence data

Your goal today is to analyze the sequencing data for you two potential mutant IPC samples - two independent colonies from your X#Z mutant - and then decide which colony to proceed with for the X#Z mutant.

  1. Use the pRSET-IPC ApE file to mark and/or note down the expected location of your mutation before proceeding.
    • You can simply compare to your annotation of the IPC alone ApE file that you prepared on Day 1 of the module.
    • You may also find it helpful to generate another ApE file with only the CaM portion of IPC and use this when you assess the Genewiz sequencing results.
  2. Your sequencing data from Genewiz is available at this link.
    • Choose the "Login" link and then use "nllyell@mit.edu" and "be20109" to access your results.
    • At the bottom right should be a link to download your sequencing results.
      • TR section: click on the Tracking Number 10-324382914 (Order Date 02-18-2016) and Tracking Number 10-324581564 (Order Date 02/21/2016)
      • WF section: click on the Tracking Number 10-324426168 (Order Date 02-19-2016) and Tracking Number 10-324584038 (Order Date 02/21/2016)
  3. The quickest way to start working with your data is to follow the "View" link under the Seq File heading. For ambiguous data, you may want to look directly at the Trace File as well.

You can align your sequencing data with a known sequence, in this case the CaM portion of inverse pericam, 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 APE and the NCBI-hosted tool are below. Please feel free to use either program...or any program with which you are familiar.

Align with ApE

  1. Open the pRSET-IPC file (linked above) or generate a CaM file for use in your alignments.
  2. Go to File and select 'New' to open a new window.
  3. Paste the sequence text from your sequencing run into the new window. 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.
    • The start and end of your sequencing may have several Ns. In this case it is best to omit these Ns by pasting only the 'good' sequence that is flanked by the ambiguous sequence.
  4. Go to Tools and select 'Align Two Sequences...'
    • In one drop-down window choose the pRSET-IPC or CaM file and in the second drop-down window choose the new file that contains the Genewiz sequence you copied and pasted in step #3.
    • Be sure to consider whether you want to compare the reverse-complement of the Genewiz sequence and, if appropriate, check the box to the right of the drop-down window. If you are unsure if this box should be checked, ask the teaching faculty.
  5. Click 'OK' and a new window should open with the sequences aligned. Matches will be shown by vertical lines between the aligned sequences. You should see a long stream of matches. If your point mutation is present, then in this stream of matches the 1 mismatched basepair should be highlighted in red.
  6. Carefully examine the sequence to see if your mutation was incorporated.
  7. You should save a screenshot of each alignment and attach them to your notebook.
  8. Follow the above steps to examine all of your sequencing results. Remember: you used a forward and a reverse primer to interrogate both potentially mutated plasmids.

If both colonies for your mutant have the correct sequence, choose one to use for the protein purification step. If only one is correct, then this is the one you will use next time. If neither of your plasmids carry the appropriate mutation, talk to the teaching faculty.

Align with "bl2seq" from NCBI

  1. The "nucleotide BLAST" alignment program can be accessed through the NCBI BLAST page or directly from this link. The default settings should be fine.
  2. Paste the sequence text from your sequencing run into the "Query" 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.
    • The start and end of your sequencing may have several Ns. In this case it is best to omit these Ns by pasting only the 'good' sequence that is flanked by the ambiguous sequence.
  3. Paste the pRSET-IPC or CaM sequence into the "Subject" box.
  4. Click on the BLAST button. 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 ~200 bp 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 1 missing line indicating your mutant codon should stand out. If it doesn't, use the numbering or Find tool to locate the appropriate codon.
  5. Carefully examine the sequence to see if your mutation was incorporated.
  6. You should save a screenshot of each alignment and attach them to your notebook.
  7. Follow the above steps to examine all of your sequencing results. Remember: you used a forward and a reverse primer to interrogate both potentially mutated plasmids.

If both colonies for your mutant have the correct sequence, choose one to use for the protein purification step. If only one is correct, then this is the one you will use next time. If neither of your plasmids carry the appropriate mutation, talk to the teaching faculty.

Part 3: Observe mutant colonies

Last time you transformed BL21(DE3)pLysS cells with three different plasmids (two candidates for the X#Z mutant, and one wild-type IPC); you also performed a no-DNA control transformation. Count the number of colonies on each plate and record the values in your notebook.

Part 4: Cell observation and collection

  1. After 2.5 hours, you will pour 1.5 mL from each tube (from Part 1) into a labeled eppendorf. Save the other 3 mL!
  2. First, measure the OD600 values of the three +IPTG samples, according to Part 5 of today's protocol.
  3. Spin the 1.5 mL +IPTG samples for 1 minute at maximum speed. Save the other 3 mL!
  4. Aspirate the supernatant from each eppendorf, using a fresh yellow pipet tip on the end of the glass pipet each time.
  5. Observe the color of each of your pellets and record this observation in your notebook. If the wild-type and both mutant pellets all appear yellow-greenish to the eye, proceed as follows:
    • Do NOT toss the rest of the liquid cultures.
    • Next, pour 1.5 mL more of the relevant liquid culture on top of each pellet, spin again, and aspirate the supernatant.
    • The last 1.5 mL of culture may be aspirated in your vacuum flask, to be later bleached and discarded.
  6. If one or more of your pellets are white or only dimly colored, please ask one of the teaching staff to show you the room temperature rotary shaker. 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 prepare to 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 the end of today, you should have six samples (3 -IPTG no-induction controls and 3 post-induction samples, 1 of each for both X#Z mutants and wild type). Measure the OD600 of a 1:10 dilution of cells for each finished sample, and write this number down in your notebook and on today's Discussion page. Then spin down the cells and aspirate the supernatant. Give the cell pellets to the teaching faculty; they will be stored frozen at -20 °C. (Be sure to make a 2X pellet for the +IPTG samples.)

Reagent list

  • IPTG (isopropyl β-D-1-thiogalactoside), 0.1 M

Navigation links

Next day: Purify protein

Previous day: Prepare expression system and evaluate DNA