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

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==Introduction==
 
==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.
+
In the previous laboratory session, you reviewed the mutations that were generated in IPC to create variants. The goal of this was to change calcium binding of IPC by affecting either affinity or cooperativity. Today you will learn how IPC and the IPC variants were expressed and purified, then you will evaluate the success of the protein purification procedure.
  
[[Image:Sp16 M1D4 pRSET vector map.png|thumb|right|350px|'''Vector map of pRSET modified from Invitrogen manual.''']]
+
[[Image:Sp16 M1D4 pRSET vector map.png|thumb|right|350px|'''Schematic of pRSET expression plasmid.''' Modified from Invitrogen manual.]] The genetic sequences that encode the IPC protein and IPC variant proteins are maintained within the pRSET expression vector (recall the cloning exercise from M3D1!).  This expression vector contains several features that are important to the expression and purification of IPC and the IPC variants. To enable selection of bacterial cells that carry pRSET_IPC, an antibiotic cassette, specifically an ampicillin marker, is included on the vector.  The features most relevant to protein expression and purification are highlighted in the schematic to the right.  The T7 promoter drives expression of the gene that encodes IPC (or IPC variant).  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. 
  
The bacterial expression vector we are using, [http://tools.invitrogen.com/content/sfs/manuals/prset_man.pdf pRSET], encodes many features that make it ideal for our research purposesTo 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 purificationIn the image to the right a schematic representation of these features is shownThe 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 includedThe 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.
+
There are some similarities between the expression system used to purify TDP43-RRM12 in Mod 2 and the system we will use for IPCFirst, in both expression systems IPTG is used to induce protein productionAs a review, IPTG is a lactose analog that induces expression by binding to the LacI repressor.  When bound to IPTG, the LacI repressor is not able to bind to the ''lac'' operator sequence and transcription occurs unimpededFor more details please review to the [[20.109(S21):M2D1#Introduction |M2D1 Introduction]]! Another similarity is that a 6His tag is used and 6His-tagged IPC and IPC variants will purified using column affinity as shown in the image belowThere are also several differences between the expression systems for TDP43-RRM12 and IPC.  As you read through the exercises below, consider how these steps are different from those used previously.
  
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<sup>-</sup>, ''omp''T ''hsd''SB (r<sub>B</sub><sup>-</sup> m<sub>B</sub><sup>-</sup>) ''gal dcm'' (DE3) pLysS (Cam<sup>R</sup>). In BL21(DE3), T7RNAP is associated with a ''lac'' construct. Constitutively expressed ''lac'' repressor (''lac''I 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 &beta;-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.
+
[[Image:Fa20 M2D1 protein purification.png|thumb|550px|center|'''Schematic of affinity separation process.''' For purification, agarose beads (yellow) are coated with nickel (green). When cell lysate is added to the nickel-coated agarose beads, His-tagged protein of interest (blue) adheres to the beads and other proteins in the lysate (orange) are washed from the beads.]]
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+
  
[[Image:Sp16 M1D4 protein expression system.png|thumb|center|600px|'''Overview of protein expression system.''']]
+
==Protocols==
  
<br style="clear:both;"/>
+
===Part 1: Participate in Comm Lab workshop===
  
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 5&alpha; 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 10<sup>9</sup> transformed cells per microgram of DNA, but even with highly competent cells only 1 DNA molecule in about 10,000 is successfully transformed.
+
Our communication instructors, Dr. Prerna Bhargava and Dr. Sean Clarke, will join us today for a discussion on preparing a Research proposal presentation.
  
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.
+
===Part 2: Prepare protein expression system===
  
==Protocols==
+
As mentioned above, IPTG is used to induce protein production in the expression systems for TDP43-RRM12 and IPC; however, the mechanism that drives transcription of the gene that encodes the protein of interest is different.  For IPC and the IPC variants, the proteins are expressed using the BL21(DE3)pLysS strain of ''E. coli'', which has the following genotype: F<sup>-</sup>, ''omp''T ''hsd''SB (r<sub>B</sub><sup>-</sup> m<sub>B</sub><sup>-</sup>) ''gal dcm'' (DE3) pLysS (Cam<sup>R</sup>).
  
===Part 1: Participate in Comm Lab workshop===
+
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.  In BL21(DE3), T7RNAP is associated with a ''lac'' construct. Constitutively expressed ''lac'' repressor (''lac''I 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 &beta;-D-thiogalactoside). To reduce ‘leaky’ expression of the protein of interest (in our case, IPC), 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 IPC) is retained by ampicillin selection.
  
Our communication instructors, Dr. Prerna Bhargava and Dr. Sean Clarke, will join us today for a discussion on preparing a Research proposal presentation.
+
The pRSET_IPC and pRSET_IPC variants were transformed into chemically competent BL21(DE3)pLysS using heat shock as described previously. To review this method, look back at the information provided on [[20.109(S21):M1D3#Part_3:_Transform_plasmid_from_yeast_into_E._coli |M1D3]]!
  
===Part 2: Induce expression of IPC variants===
+
[[Image:Sp16 M1D4 protein expression system.png|thumb|center|600px|'''Overview of protein expression system used for IPC purification.''']]
  
===Part 3: Purify IPC variants===
+
<font color = #4a9152 >'''In your laboratory notebook,'''</font color> complete the following:
 +
*questions about expression system...
  
 +
===Part 3: Purify IPC protein===
  
 +
Though the protocol refers only to the purification of IPC, the same procedure was used to purify IPC all of the IPC variants that were assessed in this experiment.
  
 +
'''Induce expression of IPC'''
  
 +
#Inoculate 5 mL of LB media containing 50 &mu;g/mL ampicillin and 34 &mu;g/mL chloramphenicol with a colony of BL21(DE3)pLysS cells transformed with pRSET_IPC.
 +
#Incubate the culture overnight at 37 &deg;C with shaking at 220 rpm.
 +
#Dilute the overnight culture 1:10 in 50 mL of fresh LB media containing 50 &mu;g/mL ampicillin and 34 &mu;g/mL chloramphenicol.
 +
#Incubate at 37 &deg;C until the OD<sub>600</sub> = ~0.6 with shaking at 220 rpm, approximately 4 hours.
 +
#To induce IPC protein expression, add IPTG to a final concentration of 1 mM.
 +
#Incubate at 25 &deg;C with shaking at 100 rpm overnight.
 +
#To harvest the cells, centrifuge the culture at 3000 g for 15 min at 4 &deg;C.
 +
#Cell pellet was stored at -80 &deg;C until used for purification.
  
===Part 1: Prepare competent BL21(DE3)pLysS cells===
+
<font color = #4a9152 >'''In your laboratory notebook,'''</font color> complete the following:
 +
*Why is it important that both ampicillin and chloramphenicol are added to the growth media?
  
#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 OD<sub>600</sub> value of 0.4-0.8.
+
'''Lyse BL21(DE3)pLysS cells expressing pRSET_IPC'''
#Measure the OD<sub>600</sub> value of a 1:10 dilution of your cells (use a total volume of 650-700 &mu;L). 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.
+
#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 &mu;L), then add the remaining volume of CaCl<sub>2</sub> (e.g., in two steps of 650 &mu;L) and invert the tubes to mix.
+
#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.
+
#This time, resuspend each pellet in 100 μL of CaCl<sub>2</sub>, then pool the cells together in one tube.
+
#*Alternatively, resuspend the first pellet in 300 &mu;L, then use this cell solution to resuspend the next pellet, and the next.
+
#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===
+
#Obtain a 2 mL aliquot of room temperature BugBuster buffer and the induced BL21(DE3)pLysS pRSET_IPC cell pellet.
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.  
+
#*BugBuster is a bacterial lysis and protein extraction solution, which contains 0.1% bovine serum albumin and 1:200 protease inhibitor cocktail to guard against protein degradation.
 +
#Add 1:1000 of cold nuclease enzyme to the BugBuster buffer.
 +
#Add 600 &mu;L of the BugBuster with nuclease enzyme to the BL21(DE3)pLysS pRSET_IPC cell pellet.
 +
#Resuspend the cell pellet by pipetting until the solution is homogeneous.
 +
#Incubate on the nutator at room temperature for 10 minutes.
 +
#Centrifuge the lysed cell suspension for 10 minutes at maximum speed.  
 +
#Transfer the supernatant to a fresh microcentrifuge tube.
  
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.
+
'''Prepare Ni-NTA affinity column'''
  
#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).
+
It is important that all liquid waste generated in the below steps is collected in a designated waste stream due to the presence of nickel in the solution!
#Vortex the bacteria and pour ~1.5 mL of each candidate into an eppendorf tube. [[Image:Removing cells.jpg|thumb|right|200px|'''Diagram showing how to aspirate the supernatant.'''  Be careful to remove as few cells as possible.]]
+
#Balance the tubes in the microfuge, spin them at maximum speed for 2 min, and remove the supernatants with the vacuum aspirator.
+
#Pour another 1.5 mL of culture onto the pellet, and repeat the spin step.
+
#Resuspend each cell pellet in 250 &mu;L buffer P1.
+
#*Buffer P1 contains RNase so that we collect only our nucleic acid of interest, DNA.
+
#Add 250 &mu;L 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.
+
#Add 350 &mu;L 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.
+
#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.
+
#Transfer the entire supernatant to the column and centrifuge for 1 min. Discard the eluant into a tube labeled ''''Qiagen waste'.'''
+
#Add 0.5 mL PB to each column, then spin for 1 min and discard the eluant into the Qiagen waste tube.
+
#Next wash with 0.75 mL PE, with a 1 min spin step as usual. Discard the ethanol in the Qiagen waste tube.
+
#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.
+
#Add 30 &mu;L of distilled H<sub>2</sub>O 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===
+
#Gently mix the Ni-NTA His-bind resin to fully resuspend, then aliquot 400 μL of the resin into a 15 mL conical tube.
 +
#Add 1.6 mL of 1X Ni-NTA Bind Buffer to the Ni-NTA His-bind resin.
 +
#Resuspend the resin by pippeting, then centrifuge at 3300 rpm for 1 minute.
 +
#Carefully remove the supernatant and discard it in the appropriate waste stream.
  
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.
+
'''Purify TDP43-RRM12 from cell lysate'''
  
The primers you will use today are below:
+
It is important that all liquid waste generated in the below steps is collected in a designated waste stream due to the presence of imidazole in the solution!
 +
 
 +
#Add the supernatent from the cell lysis to the prepared Ni-NTA His-bond resin and carefully place on the nutator at at 4&deg;C for 30 minutes.
 +
#Centrifuge at 3300 rpm for 1 minute.
 +
#Remove the liquid above the resin and discard it in the appropriate waste stream.
 +
#Add 1 mL of 1X Ni-NTA Wash Buffer to the resin and resuspend.
 +
#Centrifuge at 3300 rpm for 1 minute.
 +
#Remove the liquid above the resin and discard it in the appropriate waste stream.
 +
#Repeat Steps #4-6.
 +
#To collect your purified protein, add 500 &mu;L of 1X Ni-NTA Elute Buffer and resupend.
 +
#Centrifuge at 3300 rpm for 1 minute.
 +
#Transfer the liquid above the resin to a fresh microcentrifuge tube.
 +
#*Please note: your protein is in the liquid at this step!
 +
#Repeat Steps #8-10.
 +
#*Transfer the liquid from the second elution to the same tube used in Step #10.
 +
#*You should have a total of 1 mL of purified protein solution.
 +
 
 +
'''Remove imidazole from purified IPC'''
 +
 
 +
Pilot experiments revealed that imidazole affects the binding curves of inverse pericams. Thus, you will further purify your protein by removing low molecular weight compounds (which includes imidazole!) using a column that removes salt, or desalts, liquid as it passing through a resin.
 +
 
 +
#Obtain a Zeba column and a 15 mL conical tube.
 +
#To prepare the Zeba column, snap off the bottom of a Zeba column and place it into a 15 mL conical tube.
 +
#Centrifuge the column at 2100 rpm for 2 minutes.
 +
#Transfer the column to a fresh 15 mL conical tube, then gently apply your ~1 mL of purified protein solution to the center of the compacted resin.
 +
#Centrifuge the column at 2100 rpm for 2 minutes.
 +
#Transfer the liquid from the 15 mL conical tube to a fresh microcentrifuge tube.
 +
#*Please note: your protein is in the liquid at this step!
 +
#From the desalted purified protein solution, aliquot the following amounts:
 +
#*Add 25 &mu;L to a fresh microcentrifuge tube for examining protein purity using SDS-PAGE.
 +
#*Add 10 &mu;L to a fresh microcentrifuge tube for examining protein concentration using microBCA.
 +
#Lastly, add a 1:100 dilution of 10% BSA to the remaining desalted purified protein solution.
 +
#*For reference, 10 &mu;L of 10% BSA would be added to 1 mL of protein solution.
 +
 
 +
===Part 4: Evaluate purified IPC===
 +
 
 +
To evaluate the purified IPC protein, we will use the same methods as when we assessed purified TDP43-RRM12: SDS-PAGE and microBCA (this is a variation of the BCA procedure that is used to measure lower protein concentrations).  To review these methods, look back at the information provided on [[20.109(S21):M2D2 |M2D2]]!
 +
 
 +
'''Assess purity using SDS-PAGE'''
 +
 
 +
[[Image:Sp21 M3D3 SDSPAGE.png|thumb|500px|center]]
 +
 
 +
'''Measure concentration using microBCA'''
 +
 
 +
#calculate concentration of total protein in each sample...
 +
 
 +
#using SDS-PAGE to estimate percentage of total protein that is IPC...
 +
 
 +
#might adding different amounts of IPC variants complicate comparisons... how... (move to next day)
 +
 
 +
#how could you change experiment such that same amounts of IPC used... (move to next day)
 +
 
 +
===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>
 
<center>
 
{| border="1"
 
{| border="1"
! Primer
+
! Sample Name       
! Sequence (5' - 3')
+
! OD<sub>600</sub>
 +
! Sample Volume (&mu;L)
 +
! Water Volume (&mu;L)
 +
! Total Volume (&mu;L)
 
|-
 
|-
| IPC_F
+
| -IPTG WT
| GTC CAG GAG CGC ACC ATC TTC
+
|
 +
|
 +
|
 +
|15
 
|-
 
|-
| IPC_R
+
| +IPTG WT
| GGC CCC AAG GGG TTA TGC
+
|  
 +
|
 +
|
 +
|15
 +
|-
 +
| -IPTG mutant
 +
|
 +
|
 +
|
 +
|15
 +
|-
 +
| +IPTG mutant
 +
|
 +
|
 +
|
 +
|15
 
|-
 
|-
 
|}
 
|}
 
</center>
 
</center>
  
Use your APE file of IPC from [http://engineerbiology.org/wiki/20.109%28S16%29:In_situ_cloning_%28Day1%29 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 &mu;L. The miniprep'd plasmid should have ~300 ng of nucleic acid/&mu;L 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 &mu;L nuclease-free water
 
* 4 &mu;L of your plasmid DNA candidate
 
* 5 &mu;L of the primer stock on the teaching bench (the stock concentration is 5 pmol / &mu;L)
 
** 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.
 
  
 +
===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>
 
<center>
 
{| border="1"
 
{| border="1"
! '''T/R'''
+
! '''Vial''' <br>
! Tubes
+
! '''Volume of diluent (mL)'''
! '''W/F'''
+
! '''Volume (mL) and source of BSA (vial)'''
 +
! '''Final BSA concentration (μg/mL)'''        
 
|-
 
|-
| Red
+
| A
| 1-4
+
| 2.25
| Red
+
| 0.25 of stock
 +
| 200
 
|-
 
|-
| Orange
+
| B
| 5-8
+
| 3.6
| Orange
+
| 0.4 of A
 +
| 20
 
|-
 
|-
| Yellow
+
| C
| 9-12
+
| 2.0
| Blue
+
| 2.0 of B
 +
| 10
 
|-
 
|-
| Green
+
| D
| 13-16
+
| 2.0
| Pink
+
| 2.0 of C
 +
| 5
 
|-
 
|-
| Blue
+
| E
| 17-20
+
| 2.0
| Purple
+
| 2.0 of D
 +
| 2.5
 
|-
 
|-
| Pink
+
| F
| 21-24
+
| 2.4
|  
+
| 1.6 of E
 +
| 1
 
|-
 
|-
| Purple
+
| G
| 25-28
+
| 2.0
|  
+
| 2.0 of F
 +
| 0.5
 
|-
 
|-
 +
| H
 +
| 4.0
 +
| 0
 +
| Blank
 
|}
 
|}
 
</center>
 
</center>
  
===Part 4: Transform BL21(DE3)pLysS with pRSET-IPC X#Z===
+
====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).
#Prewarm and dry 4 LB+Amp+Cam plates by placing them in the 37 &deg;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).
+
#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.
#When your competent cells are ready, aliquot 70 &mu;L of cells per pre-chilled eppendorf.
+
#*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.
#Add 2 &mu;L 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.
+
#*'''Prepare your WR in a 15 mL conical tube.'''
#Flick to mix the contents and leave the tubes on ice for at least 5 min.
+
#Pipet 0.5 mL of each standard prepared in Part 4A into clearly labeled 1.5 mL microcentrifuge tubes.
#Heat shock the cells on the 42 &deg;C heat block for 90 s exactly and then put on ice for 2 min.
+
#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.
#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.  
+
#Add 0.5 mL of the WR to each 0.5 mL aliquot of the standard and to your 0.5 mL protein samples.
#Incubate the tubes in the 37 &deg;C incubator for at least 30 min. This gives the antibiotic-resistance genes some time to be expressed in the transformed bacterial cells.
+
#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.
#While you are waiting, label 3 large glass test tubes with your team color and sample names.
+
#Following the incubation, the teaching faculty will use the spectrophotometer to measure the protein concentrations of your standards and your purified samples.  
#*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 cuvette filled only with water (H) will be used as a blank in the spectrophotometer.
#*The teaching faculty will use these tubes to inoculate your colonies.
+
#*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.
# 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.
+
#* Establish your standard curve by plotting OD562 for each BSA standard (B-H) vs. its concentration in &mu;g/mL.
#*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.
+
#* 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).
#*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.
+
#Plate 200 &mu;L 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. 
+
#Once the plates are done, wrap them with colored tape and incubate them in the 37 &deg;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===
+
 
+
#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 OD<sub>600</sub> values of your cells (650-700 &mu;L 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.
+
#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.
+
#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.  
+
#Return your tubes to the rotary shaker in the 37 &deg;C incubator, and note down the time.
+
#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.
+
 
+
#Use the [[Media: S12-M2-20109_pRSET-IPC.gb| 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.
+
#Your sequencing data from Genewiz is available at [http://genewiz.com 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)
+
#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'''
+
#Open the pRSET-IPC file (linked above) or generate a CaM file for use in your alignments.
+
#Go to File and select 'New' to open a new window.
+
#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.
+
#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.'''
+
#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.  
+
#Carefully examine the sequence to see if your mutation was incorporated.
+
#You should save a screenshot of each alignment and attach them to your notebook.
+
#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.
+
 
+
<b>Align with "bl2seq" from [http://www.ncbi.nlm.nih.gov/ NCBI]</b>
+
#The "nucleotide BLAST" alignment program can be accessed through the NCBI [http://www.ncbi.nlm.nih.gov/BLAST/ BLAST page] or directly from this [http://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi link]. The default settings should be fine.
+
#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.
+
#Paste the pRSET-IPC or CaM sequence into the "Subject" box.
+
#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.
+
#Carefully examine the sequence to see if your mutation was incorporated.
+
#You should save a screenshot of each alignment and attach them to your notebook.
+
#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===
+
 
+
#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!
+
#First, measure the OD<sub>600</sub> values of the three +IPTG samples, according to Part 5 of today's protocol.
+
#Spin the 1.5 mL +IPTG samples for 1 minute at maximum speed. Save the other 3 mL!
+
#Aspirate the supernatant from each eppendorf, using a fresh yellow pipet tip on the end of the glass pipet each time.
+
#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. 
+
#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 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.)
+
  
 
==Reagents list==
 
==Reagents list==
 +
*Luria-Bertani broth (LB) (from Difco)
 +
*ampicillin; stock = 100 mg/mL (from Sigma)
 +
*chloramphenicol; stock = 34 mg/mL (from Sigma)
 +
*isopropyl β-d-1-thiogalactopyranoside (IPTG) (from Sigma)
 +
*BugBuster Protein Extraction Reagent (from EMD Millipore)
 +
*6X Laemmli sample buffer (from Boston BioProducts)
 +
*4-20% polyacrylamide gels in Tris-HCl (from Bio-Rad)
 +
*TGS buffer: 5 mM Tris, 192 mM glycine, 0.1% (w/v) SDS (pH 8.3) (from Bio-Rad)
 +
*Precision Plus Dual Color Standard ladder (from Bio-Rad)
 +
**Molecular weights of ladder bands (linked [http://www.bio-rad.com/en-us/product/prestained-protein-standards?ID=a7b0f9ce-e080-4b51-ab99-4cded66497c1&WT.mc_id=170125006445&WT.srch=1&WT.knsh_id=7417aea6-506f-40cd-96ae-4b6621ba8344&gclid=Cj0KCQjwjer4BRCZARIsABK4QeXhVOAohSuOrR4OKPwHbNnBCWyi5EWgxDPWCbqp67YwI-Qk7AAmMaUaAumyEALw_wcB here]).
 +
*BioSafe Coomassie G-250 Stain (from Bio-Rad)
 +
*Protein purification supplies (from Novagen/Calbiochem):
 +
**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
 +
*Zeba Desalt Spin Columns (from Thermo Scientific)
 +
*Micro BCA Protein Assay Kit (from Thermo Scientific)
  
 
==Navigation links==
 
==Navigation links==
 
Next day: [[20.109(S21):M3D4 |Evaluate effect of mutations on IPC variants ]] <br>
 
Next day: [[20.109(S21):M3D4 |Evaluate effect of mutations on IPC variants ]] <br>
 
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Revision as of 17:57, 11 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

In the previous laboratory session, you reviewed the mutations that were generated in IPC to create variants. The goal of this was to change calcium binding of IPC by affecting either affinity or cooperativity. Today you will learn how IPC and the IPC variants were expressed and purified, then you will evaluate the success of the protein purification procedure.

Schematic of pRSET expression plasmid. Modified from Invitrogen manual.
The genetic sequences that encode the IPC protein and IPC variant proteins are maintained within the pRSET expression vector (recall the cloning exercise from M3D1!). This expression vector contains several features that are important to the expression and purification of IPC and the IPC variants. To enable selection of bacterial cells that carry pRSET_IPC, an antibiotic cassette, specifically an ampicillin marker, is included on the vector. The features most relevant to protein expression and purification are highlighted in the schematic to the right. The T7 promoter drives expression of the gene that encodes IPC (or IPC variant). 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.

There are some similarities between the expression system used to purify TDP43-RRM12 in Mod 2 and the system we will use for IPC. First, in both expression systems IPTG is used to induce protein production. As a review, IPTG is a lactose analog that induces expression by binding to the LacI repressor. When bound to IPTG, the LacI repressor is not able to bind to the lac operator sequence and transcription occurs unimpeded. For more details please review to the M2D1 Introduction! Another similarity is that a 6His tag is used and 6His-tagged IPC and IPC variants will purified using column affinity as shown in the image below. There are also several differences between the expression systems for TDP43-RRM12 and IPC. As you read through the exercises below, consider how these steps are different from those used previously.

Schematic of affinity separation process. For purification, agarose beads (yellow) are coated with nickel (green). When cell lysate is added to the nickel-coated agarose beads, His-tagged protein of interest (blue) adheres to the beads and other proteins in the lysate (orange) are washed from the beads.

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: Prepare protein expression system

As mentioned above, IPTG is used to induce protein production in the expression systems for TDP43-RRM12 and IPC; however, the mechanism that drives transcription of the gene that encodes the protein of interest is different. For IPC and the IPC variants, the proteins are expressed using the BL21(DE3)pLysS strain of E. coli, which has the following genotype: F-, ompT hsdSB (rB- mB-) gal dcm (DE3) pLysS (CamR).

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. 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, IPC), 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 IPC) is retained by ampicillin selection.

The pRSET_IPC and pRSET_IPC variants were transformed into chemically competent BL21(DE3)pLysS using heat shock as described previously. To review this method, look back at the information provided on M1D3!

Overview of protein expression system used for IPC purification.

In your laboratory notebook, complete the following:

  • questions about expression system...

Part 3: Purify IPC protein

Though the protocol refers only to the purification of IPC, the same procedure was used to purify IPC all of the IPC variants that were assessed in this experiment.

Induce expression of IPC

  1. Inoculate 5 mL of LB media containing 50 μg/mL ampicillin and 34 μg/mL chloramphenicol with a colony of BL21(DE3)pLysS cells transformed with pRSET_IPC.
  2. Incubate the culture overnight at 37 °C with shaking at 220 rpm.
  3. Dilute the overnight culture 1:10 in 50 mL of fresh LB media containing 50 μg/mL ampicillin and 34 μg/mL chloramphenicol.
  4. Incubate at 37 °C until the OD600 = ~0.6 with shaking at 220 rpm, approximately 4 hours.
  5. To induce IPC protein expression, add IPTG to a final concentration of 1 mM.
  6. Incubate at 25 °C with shaking at 100 rpm overnight.
  7. To harvest the cells, centrifuge the culture at 3000 g for 15 min at 4 °C.
  8. Cell pellet was stored at -80 °C until used for purification.

In your laboratory notebook, complete the following:

  • Why is it important that both ampicillin and chloramphenicol are added to the growth media?

Lyse BL21(DE3)pLysS cells expressing pRSET_IPC

  1. Obtain a 2 mL aliquot of room temperature BugBuster buffer and the induced BL21(DE3)pLysS pRSET_IPC cell pellet.
    • BugBuster is a bacterial lysis and protein extraction solution, which contains 0.1% bovine serum albumin and 1:200 protease inhibitor cocktail to guard against protein degradation.
  2. Add 1:1000 of cold nuclease enzyme to the BugBuster buffer.
  3. Add 600 μL of the BugBuster with nuclease enzyme to the BL21(DE3)pLysS pRSET_IPC cell pellet.
  4. Resuspend the cell pellet by pipetting until the solution is homogeneous.
  5. Incubate on the nutator at room temperature for 10 minutes.
  6. Centrifuge the lysed cell suspension for 10 minutes at maximum speed.
  7. Transfer the supernatant to a fresh microcentrifuge tube.

Prepare Ni-NTA affinity column

It is important that all liquid waste generated in the below steps is collected in a designated waste stream due to the presence of nickel in the solution!

  1. Gently mix the Ni-NTA His-bind resin to fully resuspend, then aliquot 400 μL of the resin into a 15 mL conical tube.
  2. Add 1.6 mL of 1X Ni-NTA Bind Buffer to the Ni-NTA His-bind resin.
  3. Resuspend the resin by pippeting, then centrifuge at 3300 rpm for 1 minute.
  4. Carefully remove the supernatant and discard it in the appropriate waste stream.

Purify TDP43-RRM12 from cell lysate

It is important that all liquid waste generated in the below steps is collected in a designated waste stream due to the presence of imidazole in the solution!

  1. Add the supernatent from the cell lysis to the prepared Ni-NTA His-bond resin and carefully place on the nutator at at 4°C for 30 minutes.
  2. Centrifuge at 3300 rpm for 1 minute.
  3. Remove the liquid above the resin and discard it in the appropriate waste stream.
  4. Add 1 mL of 1X Ni-NTA Wash Buffer to the resin and resuspend.
  5. Centrifuge at 3300 rpm for 1 minute.
  6. Remove the liquid above the resin and discard it in the appropriate waste stream.
  7. Repeat Steps #4-6.
  8. To collect your purified protein, add 500 μL of 1X Ni-NTA Elute Buffer and resupend.
  9. Centrifuge at 3300 rpm for 1 minute.
  10. Transfer the liquid above the resin to a fresh microcentrifuge tube.
    • Please note: your protein is in the liquid at this step!
  11. Repeat Steps #8-10.
    • Transfer the liquid from the second elution to the same tube used in Step #10.
    • You should have a total of 1 mL of purified protein solution.

Remove imidazole from purified IPC

Pilot experiments revealed that imidazole affects the binding curves of inverse pericams. Thus, you will further purify your protein by removing low molecular weight compounds (which includes imidazole!) using a column that removes salt, or desalts, liquid as it passing through a resin.

  1. Obtain a Zeba column and a 15 mL conical tube.
  2. To prepare the Zeba column, snap off the bottom of a Zeba column and place it into a 15 mL conical tube.
  3. Centrifuge the column at 2100 rpm for 2 minutes.
  4. Transfer the column to a fresh 15 mL conical tube, then gently apply your ~1 mL of purified protein solution to the center of the compacted resin.
  5. Centrifuge the column at 2100 rpm for 2 minutes.
  6. Transfer the liquid from the 15 mL conical tube to a fresh microcentrifuge tube.
    • Please note: your protein is in the liquid at this step!
  7. From the desalted purified protein solution, aliquot the following amounts:
    • Add 25 μL to a fresh microcentrifuge tube for examining protein purity using SDS-PAGE.
    • Add 10 μL to a fresh microcentrifuge tube for examining protein concentration using microBCA.
  8. Lastly, add a 1:100 dilution of 10% BSA to the remaining desalted purified protein solution.
    • For reference, 10 μL of 10% BSA would be added to 1 mL of protein solution.

Part 4: Evaluate purified IPC

To evaluate the purified IPC protein, we will use the same methods as when we assessed purified TDP43-RRM12: SDS-PAGE and microBCA (this is a variation of the BCA procedure that is used to measure lower protein concentrations). To review these methods, look back at the information provided on M2D2!

Assess purity using SDS-PAGE

Sp21 M3D3 SDSPAGE.png

Measure concentration using microBCA

  1. calculate concentration of total protein in each sample...
  1. using SDS-PAGE to estimate percentage of total protein that is IPC...
  1. might adding different amounts of IPC variants complicate comparisons... how... (move to next day)
  1. how could you change experiment such that same amounts of IPC used... (move to next day)

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 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

  • Luria-Bertani broth (LB) (from Difco)
  • ampicillin; stock = 100 mg/mL (from Sigma)
  • chloramphenicol; stock = 34 mg/mL (from Sigma)
  • isopropyl β-d-1-thiogalactopyranoside (IPTG) (from Sigma)
  • BugBuster Protein Extraction Reagent (from EMD Millipore)
  • 6X Laemmli sample buffer (from Boston BioProducts)
  • 4-20% polyacrylamide gels in Tris-HCl (from Bio-Rad)
  • TGS buffer: 5 mM Tris, 192 mM glycine, 0.1% (w/v) SDS (pH 8.3) (from Bio-Rad)
  • Precision Plus Dual Color Standard ladder (from Bio-Rad)
    • Molecular weights of ladder bands (linked here).
  • BioSafe Coomassie G-250 Stain (from Bio-Rad)
  • Protein purification supplies (from Novagen/Calbiochem):
    • 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
  • Zeba Desalt Spin Columns (from Thermo Scientific)
  • Micro BCA Protein Assay Kit (from Thermo Scientific)

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