Difference between revisions of "20.109(S21):M3D3"

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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 OD<sub>600</sub> of a 1:10 dilution of cells for each finished sample, and write this number down in your notebook and on today's [http://engineerbiology.org/wiki/Talk:20.109%28S16%29:Induce_protein_expression_%28Day5%29 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 &deg;C. (Be sure to make a 2X pellet for the +IPTG samples.)
 
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 OD<sub>600</sub> of a 1:10 dilution of cells for each finished sample, and write this number down in your notebook and on today's [http://engineerbiology.org/wiki/Talk:20.109%28S16%29:Induce_protein_expression_%28Day5%29 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 &deg;C. (Be sure to make a 2X pellet for the +IPTG samples.)
 +
 +
===Part 1: Lysis of cells producing wild-type and mutant IPC===
 +
 +
#You will be given a 2 mL aliquot of room temperature BugBuster buffer (bacterial lysis and protein extraction solution), which contains 0.1% bovine serum albumin (BSA, a stabilizer) and 1:200 protease inhibitor cocktail to guard against protein degradation.
 +
#*When you are ready to begin, add 1:1000 of cold nuclease enzyme (obtained from teaching staff) to the BugBuster solution.
 +
#Per cell pellet (4 total), add the appropriate volume of enzyme-containing BugBuster and resuspend by pipetting until the solution is relatively homogeneous.
 +
#*Resuspend -IPTG samples in 300 &mu;L, and +IPTG samples in 600 &mu;L - do you remember why?
 +
#Pipet up and down to mix.
 +
#*Be sure the sample is homogenous.  You will likely need to pipet up and down 15-20x.
 +
#Incubate the solutions (at room temperature) for 10 minutes on the nutator.
 +
#*During this incubation, you may begin the resin preparation described in Part 3.
 +
#Finally, centrifuge for 10 minutes at maximum speed and transfer supernatants to fresh tubes.
 +
#While one partner completes Part 2, the other partner can begin/continue with the resin preparation in Part 3.
 +
 +
===Part 2: Advance preparation for SDS-PAGE of protein extracts===
 +
 +
#Last time you measured the amount of cells in each of your samples (-IPTG and +IPTG of the wild-type IPC and one correct mutant). (If you ran cultures overnight, the teaching faculty measured the +IPTG samples for you and posted the results.) Look back at your measurements, and find the sample with the lowest cell concentration. Set aside 15 &mu;L of this sample for PAGE analysis in an eppendorf.
 +
#For your other three samples, you should take the amount of bacterial lysate corresponding to the same number of cells as the lowest concentration sample. For example, if the OD<sub>600</sub> of your WT -IPTG sample was 0.05, and the OD<sub>600</sub> of your WT +IPTG sample was 0.30, you would take  15 &mu;L of the -IPTG, but only 2.5 &mu;L of the +IPTG sample.
 +
#Next, add enough water so the each sample has 15 &mu;L of liquid in it. You might use the table below to guide your work.
 +
#Finally, add 3 μL of 6X sample buffer to 15 μL of each of your diluted lysates. These will be stored in the freezer until next time.
 +
 +
<center>
 +
{| border="1"
 +
! Sample Name       
 +
! OD<sub>600</sub>
 +
! Sample Volume (&mu;L)
 +
! Water Volume (&mu;L)
 +
! Total Volume (&mu;L)
 +
|-
 +
| -IPTG WT
 +
|
 +
|
 +
|
 +
|15
 +
|-
 +
| +IPTG WT
 +
|
 +
|
 +
|
 +
|15
 +
|-
 +
| -IPTG mutant
 +
|
 +
|
 +
|
 +
|15
 +
|-
 +
| +IPTG mutant
 +
|
 +
|
 +
|
 +
|15
 +
|-
 +
|}
 +
</center>
 +
 +
===Part 3: Protein purification===
 +
 +
====Part 3A: Nickel-agarose purification====
 +
 +
You will process two samples (+IPTG wild-type and +IPTG mutant IPC) according to the following procedure. <!--
 +
You should either time your spins with another group, or balance your tubes with 3-way symmetry.
 +
-->
 +
'''Keep all buffers on ice when  not in use.''' <font color = CC0066>'''All spins should be performed at 1000 rcf (3300 rpm) for 1 minute.'''</font color>
 +
 +
#The following buffers are aliquoted and located at the front bench:
 +
#*Ni-NTA His-bind resin
 +
#*1X Ni-NTA Bind Buffer (50 mM NaH<sub>2</sub>PO<sub>4</sub>, pH 8.0; 300 mM NaCl; 10 mM imidazole)
 +
#*1X Ni-NTA Wash Buffer (50 mM NaH<sub>2</sub>PO<sub>4</sub>, pH 8.0; 300 mM NaCl; 20 mM imidazole)
 +
#*1X Ni-NTA Elute Buffer (50 mM NaH<sub>2</sub>PO<sub>4</sub>, pH 8.0; 300 mM NaCl; 250 mM imidazole)
 +
#*''Note'': '''Two special waste streams''' should be created for this affinity purification procedure, (1) nickel waste for the 50% slurry, and (2) imidazole waste for the Bind, Wash, and Elute Buffers.
 +
#Gently mix the Ni-NTA His-bind resin to fully resuspend it, then distribute 400 μL of the resin to each of two 2 mL centrifuge tubes.
 +
#*Label one tube as wild type and the other as mutant.
 +
#Add 1.6 mL (2 x 800 μL) of 1X Ni-NTA Bind Buffer to the Ni-NTA His-bind resin.
 +
#*Resuspend the resin by pippeting the solution up and down several times (10-15), then centrifuge (see conditions above).
 +
#Carefully remove the supernatant and discard it in the appropriate waste stream.
 +
#Add your cleared cell lysate from Part 1 to the resin, then put your tubes on the nutator at 4&deg;C for 30 minutes.
 +
#*Be sure to add the wild type and mutant lysates to the correct tubes!
 +
#Centrifuge (see conditions above).
 +
#Remove the supernatent and discard it in the appropriate waste stream.
 +
#Add 1 mL of 1X Ni-NTA Wash Buffer to the resin.
 +
#Centrifuge (see conditions above).
 +
#Remove the supernatent and discard it in the appropriate waste stream.
 +
#Repeat Steps #8-10.
 +
#Finally, you will collect your protein. Add 500 &mu;L of Elute Buffer, resuspend, and spin as usual. '''Do not throw away the supernatant!''' Instead, transfer it to a fresh eppendorf tube, labeled “pure IPC X#Z" or “pure IPC WT.”
 +
#'''Do not throw away the resin yet either!''' Instead, repeat Step #12 one more time and add the supernatant to the sample collected in Step #12.  You'll have a total of 1 mL of pure wild-type and 1 mL of pure mutant IPC.
 +
 +
====Part 3B: Desalting====
 +
 +
We found from pilot studies that imidazole affects the binding curves of inverse pericams. Thus, you will continue purifying your proteins by removing any low molecular weight compounds.
 +
 +
#For each of your two samples, snap off the bottom of a Zeba column, place in a 15 mL conical tube, and loosen the column's cap.
 +
#In the large centrifuge in the cold room across the hall, spin your columns at 1000 rcf (which is 2100 rpm for the rotor inside this centrifuge) for 2 minutes.
 +
<!--
 +
#*The timing function on this centrifuge does not work! Bring your timer and manually turn the centrifuge off after 2 minutes. Start the timer once the rpm value approaches 2000.
 +
-->
 +
#*Because we all have to share one centrifuge, ideally spin with at least 2 other groups.
 +
#Transfer the column to a fresh 15 mL conical tube, and then gently apply your ~1 mL of protein to the center of the compacted resin.
 +
#Repeat the 2-minute spin step just as before.
 +
#Immediately after eluting your protein, transfer 10 μL of purified protein to a clean eppendorf tube for assaying protein concentrations (Part 4). Also take 15 μL to a separate eppendorf tube for SDS-PAGE analysis and add 3μL of 6X SDS-PAGE loading dye(give this tube to your instructor).
 +
#Then add a 1:100 dilution of 10% BSA to the remaining protein (10 μL of BSA for ~1 mL of protein).
 +
 +
===Part 4: Protein concentration===
 +
====Part 4A: Prepare diluted albumin (BSA) standards====
 +
#Obtain a 0.25 mL aliquot of 2.0 mg/mL albumin standard stock and a conical tube of diH<sub>2</sub>O from the front bench.
 +
#Prepare your standards according to the table below using dH<sub>2</sub>O as the diluent:
 +
#*'''Be sure to use 5 mL polystyrene tubes found on the instructors bench when preparing your standards as the volumes are too large for the microcentrifuge tubes.'''
 +
<center>
 +
{| border="1"
 +
! '''Vial''' <br>
 +
! '''Volume of diluent (mL)'''
 +
! '''Volume (mL) and source of BSA (vial)'''
 +
! '''Final BSA concentration (μg/mL)'''         
 +
|-
 +
| A
 +
| 2.25
 +
| 0.25 of stock
 +
| 200
 +
|-
 +
| B
 +
| 3.6
 +
| 0.4 of A
 +
| 20
 +
|-
 +
| C
 +
| 2.0
 +
| 2.0 of B
 +
| 10
 +
|-
 +
| D
 +
| 2.0
 +
| 2.0 of C
 +
| 5
 +
|-
 +
| E
 +
| 2.0
 +
| 2.0 of D
 +
| 2.5
 +
|-
 +
| F
 +
| 2.4
 +
| 1.6 of E
 +
| 1
 +
|-
 +
| G
 +
| 2.0
 +
| 2.0 of F
 +
| 0.5
 +
|-
 +
| H
 +
| 4.0
 +
| 0
 +
| Blank
 +
|}
 +
</center>
 +
 +
====Part 4B: Prepare Working Reagent (WR) and measuring protein concentration====
 +
#Use the following formula to calculate the volume of WR required:  (# of standards + # unknowns) * 1.1 = total volume of WR (in mL).
 +
#Prepare the calculated volume of WR by mixing the Micro BCA Reagent MA, Reagent MB, and Reagent MC such that 50% of the total volume is MA, 48% is MB, and 2% is MC.
 +
#*For example, if your calculated total volume of WR is 100 mL, then mix 50 mL of MA, 48 mL of MB, and 2 mL of MC.
 +
#*'''Prepare your WR in a 15 mL conical tube.'''
 +
#Pipet 0.5 mL of each standard prepared in Part 4A into clearly labeled 1.5 mL microcentrifuge tubes.
 +
#Prepare your protein samples by adding 990 μL of dH<sub>2</sub>O to your 10 μL aliquot of purified protein, for a final volume of 1 mL in clearly labeled 1.5 mL microcentrifuge tubes.
 +
#Add 0.5 mL of the WR to each 0.5 mL aliquot of the standard and to your 0.5 mL protein samples.
 +
#Cap your tubes and incubate at 60&deg;C in the water bath for 1 hour. During this time download the sample data on the Discussion page to practice estimating protein concentration of your samples.
 +
#Following the incubation, the teaching faculty will use the spectrophotometer to measure the protein concentrations of your standards and your purified samples.
 +
#* The cuvette filled only with water (H) will be used as a blank in the spectrophotometer.
 +
#*The absorbance at 562 nm for each solution will be measured and the results will be posted to today's [http://engineerbiology.org/wiki/Talk:20.109%28S16%29:Purify_protein_%28Day6%29 Discussion] page.
 +
#* Establish your standard curve by plotting OD562 for each BSA standard (B-H) vs. its concentration in &mu;g/mL.
 +
#* Use the standard curve in its linear range (0.5 - 20 &mu;g/mL), and its linear regression in Excel, to determine the protein concentration of each unknown sample (wild-type and mutant IPC).
  
 
==Reagents list==
 
==Reagents list==

Revision as of 22:12, 2 February 2021

20.109(S21): Laboratory Fundamentals of Biological Engineering

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Spring 2021 schedule        FYI        Assignments        Homework        Communication |        Accessibility

       M1: Antibody engineering        M2: Drug discovery        M3: Protein engineering       


Introduction

Now that you have prepared DNA encoding your mutant inverse pericams, we need to produce the proteins so we can assess the affinity and cooperativity of your new protein. Since the last time you were here, your oh-so devoted teaching staff transformed competent bacteria (strain NEB 5α) with your SDM product. Successfully transformed NEB 5α bacteria grew into colonies on ampicillin-containing plates, and two colonies were picked to grow in liquid cultures. The liquid culture of E. coli serves as a plasmid-generating factory. Today you will purify the plasmids carrying the hopefully mutated inverse pericam gene using a Qiagen mini-prep kit. To ensure that the SDM reaction was successful, and therefore the IPC gene was mutated, you will prepare the purified DNA for sequencing analysis. Though the NEB 5α cell strain is able to replicate the inverse pericam plasmid DNA, it cannot produce the inverse pericam protein. Therefore, you will transform your IPC mutant plasmids into a new bacterial system, BL21(DE3)pLysS, that can produce the protein for later analysis.

Vector map of pRSET modified from Invitrogen manual.

The bacterial expression vector we are using, pRSET, encodes many features that make it ideal for our research purposes. To enable selection of bacterial cells that carry the plasmid, an antibiotic cassette (specifically, an ampicillin marker) is included on the vector. Several features are included to promote protein expression and purification. In the image to the right a schematic representation of these features is shown. The T7 promoter drives expression of the gene that encodes IPC (or your mutated IPC). To ensure that the transcript is translated into a protein, a Ribosome Binding Site (RBS) is included. The ATG sequence serves as the transcriptional start and the 6xHis represents the six-histidine residue tag that is used for protein purification via affinity chromatography.

As mentioned above, pRSET encodes the bacteriophage T7 promoter, which is active only in the presence of T7 RNA polymerase (T7RNAP), an enzyme that therefore must be expressed by the bacterial strain used to make the protein of interest. We will use the BL21(DE3)pLysS strain, which has the following genotype: F-, ompT hsdSB (rB- mB-) gal dcm (DE3) pLysS (CamR). In BL21(DE3), T7RNAP is associated with a lac construct. Constitutively expressed lac repressor (lacI gene) blocks expression from the lac promoter; thus, the polymerase will not be produced except in the presence of repressor-binding lactose or a small-molecule lactose analogue such as IPTG (isopropyl β-D-thiogalactoside). To reduce ‘leaky’ expression of the protein of interest (in our case, inverse pericam), the pLysS version of BL21(DE3) contains T7 lysozyme, which inhibits basal transcription of T7RNAP. This gene is retained by chloramphenicol selection, while the pRSET plasmid itself (and thus inverse pericam) is retained by ampicillin selection.

Overview of protein expression system.


After completing mini-preps to isolate your plasmid DNA today (two mutant pRSET-IPC candidates), you will prepare the DNA for sequencing analysis, as well as use it immediately for transformation. In order to transform BL21(DE3)pLysS cells with your mutant IPC plasmids, you will first have to make the cells competent, i.e., able to efficiently take up foreign DNA. With the NEB 5α strain, we used commercially available competent cells that did not need further treatment prior to DNA addition. Today, you will make chemically competent cells yourself using calcium chloride, then incubate them with plasmid DNA and heat shock them as before prior to plating. Whether prepared by a company or by you, remember that competent cells are extremely fragile and should be handled gently, i.e. kept cold and not vortexed. Bacterial transformation is efficient enough for most lab purposes, resulting in as many as 109 transformed cells per microgram of DNA, but even with highly competent cells only 1 DNA molecule in about 10,000 is successfully transformed.

After today's lab session, the teaching staff will pick colonies and set up liquid overnight cultures from your transformed BL21(DE3)pLysS cells. Next time, you will add IPTG to these liquid cultures to induce expression of your mutant proteins, which you will then isolate and characterize.

Protocols

Part 1: Participate in Comm Lab workshop

Our communication instructors, Dr. Prerna Bhargava and Dr. Sean Clarke, will join us today for a discussion on preparing a Research proposal presentation.

Part 2: Induce expression of IPC variants

Part 3: Purify IPC variants

Part 1: Prepare competent BL21(DE3)pLysS cells

  1. Pick up one 5 mL tube of BL21(DE3)pLysS cells. These cells should be in or near the early- or mid-log phase of growth, which is indicated by an OD600 value of 0.4-0.8.
  2. Measure the OD600 value of a 1:10 dilution of your cells (use a total volume of 650-700 μL). If the cells are not yet dense enough, return them to the rotary shaker in the incubator. Remember to balance with another tube! As a rule, your cells should double every 20-30 minutes.
  3. Once your cells have reached the appropriate growth phase, aliquot them into 3 eppendorf tubes each containing 1.5 mL culture volume. Spin for 1 min at max speed (~16,000 rcf/13,000 rpm), aspirate the supernatants, and resuspend in 1.5 mL of ice-cold calcium chloride (100 mM). Note: you can balance these tubes in the centrifuge with three-way symmetry.
    • You may find it easiest to resuspend the cells in a small volume first (say, 200 μL), then add the remaining volume of CaCl2 (e.g., in two steps of 650 μL) and invert the tubes to mix.
  4. Spin again for 1 min. The resultant pellets should occur as streaks down the side of the eppendorf tube, so be very careful not to disturb the cells when aspirating.
  5. This time, resuspend each pellet in 100 μL of CaCl2, then pool the cells together in one tube.
    • Alternatively, resuspend the first pellet in 300 μL, then use this cell solution to resuspend the next pellet, and the next.
  6. Incubate on ice for 1 h. You can work on Parts 2, 3 and 5 of today's protocols now, as well as assemble the materials for Part 4. Among other things, be sure to label four eppendorfs and pre-chill them on ice. The labels should indicate a (-) no DNA control, a (+) wild-type IPC transformation control (with minipreps prepared and vetted by the teaching staff), and your two mutant candidate transformations (X#Z -1 and -2).

Part 2: Mini-prep pRSET-IPC X#Z

The procedure for DNA isolation using small volumes is commonly termed "mini-prep," which distinguishes it from a “maxi-prep” that involves a larger volume of cells and additional steps of purification. The overall goal of each prep is the same -- to separate the plasmid DNA from the chromosomal DNA and cellular debris. In the traditional mini-prep protocol, the media is removed from the cells by centrifugation. The cells are resuspended in a solution that contains Tris to buffer the cells and EDTA to bind divalent cations in the lipid bilayer, thereby weakening the cell envelope. A solution of sodium hydroxide and sodium dodecyl sulfate (SDS) is then added. The base denatures the DNA, both chromosomal and plasmid, while the detergent dissolves the cellular proteins and lipids. The pH of the solution is returned to neutral by adding a mixture of acetic acid and potassium acetate. At neutral pH the SDS precipitates from solution, carrying with it the dissolved proteins and lipids. In addition, the DNA strands renature at neutral pH. The chromosomal DNA, which is much longer than the plasmid DNA, renatures as a tangle that gets trapped in the SDS precipitate. The plasmid DNA renatures normally and stays in solution. Thus plasmid DNA got effectively separated from chromosomal DNA and proteins and lipids of the cell.

Today you will use a kit that relies on a column to collect the renatured plasmid DNA. The silica gel column interacts with the DNA while allowing contaminants to pass through the column. This interaction is aided by chaotropic salts and ethanol, which are added in the buffers. The ethanol dehydrates the DNA backbone allowing the chaotropic salts to form salt bridge between the silica and the DNA.

  1. Pick up your two cultures, which are growing in test tubes labeled with your team color. Label two eppendorf tubes to reflect your samples (X#Z 1 and 2).
  2. Vortex the bacteria and pour ~1.5 mL of each candidate into an eppendorf tube.
    Diagram showing how to aspirate the supernatant. Be careful to remove as few cells as possible.
  3. Balance the tubes in the microfuge, spin them at maximum speed for 2 min, and remove the supernatants with the vacuum aspirator.
  4. Pour another 1.5 mL of culture onto the pellet, and repeat the spin step.
  5. Resuspend each cell pellet in 250 μL buffer P1.
    • Buffer P1 contains RNase so that we collect only our nucleic acid of interest, DNA.
  6. Add 250 μL of buffer P2 to each tube, and mix by inversion until the suspension is homogeneous. About 4-6 inversions of the tube should suffice. You may incubate here for up to 5 minutes, but not more.
    • Buffer P2 contains sodium hydroxide for lysing.
  7. Add 350 μL buffer N3 to each tube, and mix immediately by inversion (4-10 times).
    • Buffer N3 contains acetic acid, which will cause the chromosomal DNA to messily precipitate; the faster you invert, the more homogeneous the precipitation will be.
    • Buffer N3 also contains a chaotropic salt in preparation for the silica column purification.
  8. Centrifuge for 10 minutes at maximum speed. Note that you will be saving the supernatant after this step.
    • Meanwhile, prepare 2 labeled QIAprep columns, one for each candidate clone, and 2 trimmed eppendorf tubes for the final elution step.
  9. Transfer the entire supernatant to the column and centrifuge for 1 min. Discard the eluant into a tube labeled 'Qiagen waste'.
  10. Add 0.5 mL PB to each column, then spin for 1 min and discard the eluant into the Qiagen waste tube.
  11. Next wash with 0.75 mL PE, with a 1 min spin step as usual. Discard the ethanol in the Qiagen waste tube.
  12. After removing the PE, spin the mostly dry column for 1 more minute.
    • It is important to remove all traces of ethanol, as they may interfere with subsequent work with the DNA.
  13. Add 30 μL of distilled H2O pH ~8 to the top center of the column, wait 1 min, and then spin 1 min to collect your DNA.

Part 3: Prepare DNA for sequencing reactions

Just like amplification reactions require a primer for initiation, primers are also needed for sequencing reactions. Legible readout of the gene typically begins about 40-50 bp downstream of the primer site, and continues for ~1000 bp at most. Thus, multiple primers must be used to fully view genes > 1 kbp in size. How many basepairs long is inverse pericam? Luckily, we only care about the back end of IPC, i.e. the part containing calmodulin, and therefore only need two primers to confirm our mutations: one primer will sequence in the forward direction and the second in the reverse direction to ensure complete coverage of the CaM gene.

The primers you will use today are below:

Primer Sequence (5' - 3')
IPC_F GTC CAG GAG CGC ACC ATC TTC
IPC_R GGC CCC AAG GGG TTA TGC

Use your APE file of IPC from M1D1 to determine where these primers anneal within the sequence. How many basepairs upstream and downstream of CaM do IPC_F and IPC_R, respectively, anneal?

The recommended composition of sequencing reactions is ~800 ng of plasmid DNA and 25 pmoles of sequencing primer in a final volume of 15 μL. The miniprep'd plasmid should have ~300 ng of nucleic acid/μL but that will be a mixture of RNA and DNA, so we will estimate the amount appropriate for our reactions.

Because you will examine the sequence of your potential mutants with both the IPC_F and IPC_R primers, you will need to prepare two reactions for each candidate. Thus you will have a total of four sequencing reactions. For each reaction, combine the following reagents directly in the appropriate tube within the 8-PCR-tube strip, as noted in the table below:

  • 6 μL nuclease-free water
  • 4 μL of your plasmid DNA candidate
  • 5 μL of the primer stock on the teaching bench (the stock concentration is 5 pmol / μL)
    • Please add the forward primer to the odd numbered tubes and the reverse primer to the even numbered tubes (i.e. tube #1 contains mutant #1 plasmid DNA and IPC_F primer, tube #2 contains mutant #1 plasmid DNA and IPC_R primer, etc).

The side of each tube is numerically labeled and you should use only the four tubes assigned to your group. The teaching faculty will turn in the strips at the Genewiz company drop-off box for sequencing.

T/R Tubes W/F
Red 1-4 Red
Orange 5-8 Orange
Yellow 9-12 Blue
Green 13-16 Pink
Blue 17-20 Purple
Pink 21-24
Purple 25-28

Part 4: Transform BL21(DE3)pLysS with pRSET-IPC X#Z

  1. Prewarm and dry 4 LB+Amp+Cam plates by placing them in the 37 °C incubator, media side up with the lids ajar. You will perform one transformation for each of your four samples (1 wild-type IPC, 2 mutants, and 1 no-DNA negative-control transformation).
  2. When your competent cells are ready, aliquot 70 μL of cells per pre-chilled eppendorf.
  3. Add 2 μL of the appropriate DNA to each tube. Remember, you are testing plasmid DNA that was prepared from two different colonies for your X#Z mutant, along with DNA from a colony that amplified the pRSET-IPC wild type. You will also perform a no DNA control.
  4. Flick to mix the contents and leave the tubes on ice for at least 5 min.
  5. Heat shock the cells on the 42 °C heat block for 90 s exactly and then put on ice for 2 min.
  6. Move the samples to a rack on your bench, add 0.5 mL of LB media to each one, and invert each tube to mix.
  7. Incubate the tubes in the 37 °C incubator for at least 30 min. This gives the antibiotic-resistance genes some time to be expressed in the transformed bacterial cells.
  8. While you are waiting, label 3 large glass test tubes with your team color and sample names.
    • Mix 10 mL LB broth with ampicillin and chloramphenicol, for final antibiotics concentrations of 100 ug/mL and 34 ug/mL, respectively. Aliquot 3 mL of this mixture per culture tube.
    • The teaching faculty will use these tubes to inoculate your colonies.
  9. Also prepare 4 eppendorf tubes containing 180 μL of LB each. You will use these to dilute your transformed cells 1:10 when you retrieve them from the incubator.
    • If you label these tubes with stickers rather than directly on the cap, you can then transfer each sticker to the appropriate plate as you go, saving one labeling step.
    • Note that we are reducing the cell concentration because miniprep DNA is much more concentrated than the DNA resulting from mutagenesis; it also does not require repair, further increasing the transformation efficiency.
  10. Plate 200 μL of each (1:10 diluted) transformation mix on a LB+Amp+Cam plate.
    • Safety reminder: After dipping the glass spreader in the ethanol jar, then pass it through the flame of the alcohol burner just long enough to ignite the ethanol. After letting the ethanol burn off, the spreader may still be very hot, and it is advisable to tap it gently on a portion of the agar plate without cells in order to equilibrate it with the agar.
  11. Once the plates are done, wrap them with colored tape and incubate them in the 37 °C incubator overnight. One of the teaching faculty will remove them from the incubator and set up liquid cultures for you to use next time.

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.)

Part 1: Lysis of cells producing wild-type and mutant IPC

  1. You will be given a 2 mL aliquot of room temperature BugBuster buffer (bacterial lysis and protein extraction solution), which contains 0.1% bovine serum albumin (BSA, a stabilizer) and 1:200 protease inhibitor cocktail to guard against protein degradation.
    • When you are ready to begin, add 1:1000 of cold nuclease enzyme (obtained from teaching staff) to the BugBuster solution.
  2. Per cell pellet (4 total), add the appropriate volume of enzyme-containing BugBuster and resuspend by pipetting until the solution is relatively homogeneous.
    • Resuspend -IPTG samples in 300 μL, and +IPTG samples in 600 μL - do you remember why?
  3. Pipet up and down to mix.
    • Be sure the sample is homogenous. You will likely need to pipet up and down 15-20x.
  4. Incubate the solutions (at room temperature) for 10 minutes on the nutator.
    • During this incubation, you may begin the resin preparation described in Part 3.
  5. Finally, centrifuge for 10 minutes at maximum speed and transfer supernatants to fresh tubes.
  6. While one partner completes Part 2, the other partner can begin/continue with the resin preparation in Part 3.

Part 2: Advance preparation for SDS-PAGE of protein extracts

  1. Last time you measured the amount of cells in each of your samples (-IPTG and +IPTG of the wild-type IPC and one correct mutant). (If you ran cultures overnight, the teaching faculty measured the +IPTG samples for you and posted the results.) Look back at your measurements, and find the sample with the lowest cell concentration. Set aside 15 μL of this sample for PAGE analysis in an eppendorf.
  2. For your other three samples, you should take the amount of bacterial lysate corresponding to the same number of cells as the lowest concentration sample. For example, if the OD600 of your WT -IPTG sample was 0.05, and the OD600 of your WT +IPTG sample was 0.30, you would take 15 μL of the -IPTG, but only 2.5 μL of the +IPTG sample.
  3. Next, add enough water so the each sample has 15 μL of liquid in it. You might use the table below to guide your work.
  4. Finally, add 3 μL of 6X sample buffer to 15 μL of each of your diluted lysates. These will be stored in the freezer until next time.
Sample Name OD600 Sample Volume (μL) Water Volume (μL) Total Volume (μL)
-IPTG WT 15
+IPTG WT 15
-IPTG mutant 15
+IPTG mutant 15

Part 3: Protein purification

Part 3A: Nickel-agarose purification

You will process two samples (+IPTG wild-type and +IPTG mutant IPC) according to the following procedure. Keep all buffers on ice when not in use. All spins should be performed at 1000 rcf (3300 rpm) for 1 minute.

  1. The following buffers are aliquoted and located at the front bench:
    • Ni-NTA His-bind resin
    • 1X Ni-NTA Bind Buffer (50 mM NaH2PO4, pH 8.0; 300 mM NaCl; 10 mM imidazole)
    • 1X Ni-NTA Wash Buffer (50 mM NaH2PO4, pH 8.0; 300 mM NaCl; 20 mM imidazole)
    • 1X Ni-NTA Elute Buffer (50 mM NaH2PO4, pH 8.0; 300 mM NaCl; 250 mM imidazole)
    • Note: Two special waste streams should be created for this affinity purification procedure, (1) nickel waste for the 50% slurry, and (2) imidazole waste for the Bind, Wash, and Elute Buffers.
  2. Gently mix the Ni-NTA His-bind resin to fully resuspend it, then distribute 400 μL of the resin to each of two 2 mL centrifuge tubes.
    • Label one tube as wild type and the other as mutant.
  3. Add 1.6 mL (2 x 800 μL) of 1X Ni-NTA Bind Buffer to the Ni-NTA His-bind resin.
    • Resuspend the resin by pippeting the solution up and down several times (10-15), then centrifuge (see conditions above).
  4. Carefully remove the supernatant and discard it in the appropriate waste stream.
  5. Add your cleared cell lysate from Part 1 to the resin, then put your tubes on the nutator at 4°C for 30 minutes.
    • Be sure to add the wild type and mutant lysates to the correct tubes!
  6. Centrifuge (see conditions above).
  7. Remove the supernatent and discard it in the appropriate waste stream.
  8. Add 1 mL of 1X Ni-NTA Wash Buffer to the resin.
  9. Centrifuge (see conditions above).
  10. Remove the supernatent and discard it in the appropriate waste stream.
  11. Repeat Steps #8-10.
  12. Finally, you will collect your protein. Add 500 μL of Elute Buffer, resuspend, and spin as usual. Do not throw away the supernatant! Instead, transfer it to a fresh eppendorf tube, labeled “pure IPC X#Z" or “pure IPC WT.”
  13. Do not throw away the resin yet either! Instead, repeat Step #12 one more time and add the supernatant to the sample collected in Step #12. You'll have a total of 1 mL of pure wild-type and 1 mL of pure mutant IPC.

Part 3B: Desalting

We found from pilot studies that imidazole affects the binding curves of inverse pericams. Thus, you will continue purifying your proteins by removing any low molecular weight compounds.

  1. For each of your two samples, snap off the bottom of a Zeba column, place in a 15 mL conical tube, and loosen the column's cap.
  2. In the large centrifuge in the cold room across the hall, spin your columns at 1000 rcf (which is 2100 rpm for the rotor inside this centrifuge) for 2 minutes.
    • Because we all have to share one centrifuge, ideally spin with at least 2 other groups.
  3. Transfer the column to a fresh 15 mL conical tube, and then gently apply your ~1 mL of protein to the center of the compacted resin.
  4. Repeat the 2-minute spin step just as before.
  5. Immediately after eluting your protein, transfer 10 μL of purified protein to a clean eppendorf tube for assaying protein concentrations (Part 4). Also take 15 μL to a separate eppendorf tube for SDS-PAGE analysis and add 3μL of 6X SDS-PAGE loading dye(give this tube to your instructor).
  6. Then add a 1:100 dilution of 10% BSA to the remaining protein (10 μL of BSA for ~1 mL of protein).

Part 4: Protein concentration

Part 4A: Prepare diluted albumin (BSA) standards

  1. Obtain a 0.25 mL aliquot of 2.0 mg/mL albumin standard stock and a conical tube of diH2O from the front bench.
  2. Prepare your standards according to the table below using dH2O as the diluent:
    • Be sure to use 5 mL polystyrene tubes found on the instructors bench when preparing your standards as the volumes are too large for the microcentrifuge tubes.
Vial
Volume of diluent (mL) Volume (mL) and source of BSA (vial) Final BSA concentration (μg/mL)
A 2.25 0.25 of stock 200
B 3.6 0.4 of A 20
C 2.0 2.0 of B 10
D 2.0 2.0 of C 5
E 2.0 2.0 of D 2.5
F 2.4 1.6 of E 1
G 2.0 2.0 of F 0.5
H 4.0 0 Blank

Part 4B: Prepare Working Reagent (WR) and measuring protein concentration

  1. Use the following formula to calculate the volume of WR required: (# of standards + # unknowns) * 1.1 = total volume of WR (in mL).
  2. Prepare the calculated volume of WR by mixing the Micro BCA Reagent MA, Reagent MB, and Reagent MC such that 50% of the total volume is MA, 48% is MB, and 2% is MC.
    • For example, if your calculated total volume of WR is 100 mL, then mix 50 mL of MA, 48 mL of MB, and 2 mL of MC.
    • Prepare your WR in a 15 mL conical tube.
  3. Pipet 0.5 mL of each standard prepared in Part 4A into clearly labeled 1.5 mL microcentrifuge tubes.
  4. Prepare your protein samples by adding 990 μL of dH2O to your 10 μL aliquot of purified protein, for a final volume of 1 mL in clearly labeled 1.5 mL microcentrifuge tubes.
  5. Add 0.5 mL of the WR to each 0.5 mL aliquot of the standard and to your 0.5 mL protein samples.
  6. Cap your tubes and incubate at 60°C in the water bath for 1 hour. During this time download the sample data on the Discussion page to practice estimating protein concentration of your samples.
  7. Following the incubation, the teaching faculty will use the spectrophotometer to measure the protein concentrations of your standards and your purified samples.
    • The cuvette filled only with water (H) will be used as a blank in the spectrophotometer.
    • The absorbance at 562 nm for each solution will be measured and the results will be posted to today's Discussion page.
    • Establish your standard curve by plotting OD562 for each BSA standard (B-H) vs. its concentration in μg/mL.
    • Use the standard curve in its linear range (0.5 - 20 μg/mL), and its linear regression in Excel, to determine the protein concentration of each unknown sample (wild-type and mutant IPC).

Reagents list

  • QIAprep Spin Miniprep Kit reagents
  • Sequencing primers (concentration = 5 pmol/μL)
  • 100 mM CaCl2, sterile
  • LB (Luria-Bertani broth)
    • 1% Tryptone
    • 0.5% Yeast Extract
    • 1% NaCl
    • autoclaved for sterility
  • Ampicillin stock: 100 mg/mL, aqueous, sterile-filtered, store at +4 °C
  • Chloramphenicol stock: 34 mg/mL in ethanol, store at -20 °C
  • LB+AMP+CAM plates
    • LB with 1.5% agar and 100 μg/mL ampicillin and 34 μg/mL chloramphenicol


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

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