Team:BYU Provo/Notebook/CholeraDetection/SummerExp/Period4/Dailylog

From 2013.igem.org

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Dr. Grose helped us devise an experiment to answer the questions we posed in the last entry. We have taken TT9901, TT9907 and TT25281 and transformed pIG87 (with CRO) into the strains via electroporation, and done the same procedure with its empty backbone plasmid (pIG12). We’ve grown the transformed bacteria in overnights at 30 degrees (previously we had been doing our molecular work at 37 degrees, which could be hot enough to prematurely stress lambda into lysis), and then performed a top agar assay that has included the following variables: top agar plate assays with/without amp, with/without arabinose, and with/without H202, for both our CRO+ and CRO- strains. We thought that the comparing the combinations of plates tell us what is responsible for our previous no-plaque observation.
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Set up 40ul Digests: 1. pIG89, XhoI, XbaI, NEB4 2. pIG78, XhoI, XbaI, BamHI, NEB4. 3. pIG78, HindIII, XbaI, BamHI, NEB4. 4. pIG10, HindIII, XbaI, NEB4. All the digests worked great. The addition of BamHI helped resolve the problem of the bands being to close together. Collected a slice of each from a low melt gel.  
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Well, once again, our plates we plaque-less.  Not one plaque on any of the 21 top agar lawns, with or without H202, with or without arabinose, CRO, etc.
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The plot thickens.  We’re double-checking our frozen strains to ensure they can still be induced to lysis.
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Set up 40ul Digests: 1. pIG89, XhoI, XbaI, NEB4 2. pIG78, XhoI, XbaI, BamHI, NEB4. 3. pIG78, HindIII, XbaI, BamHI, NEB4. 4. pIG10, HindIII, XbaI, NEB4. All the digests worked great. The addition of BamHI helped resolve the problem of the bands being to close together. Collected a slice of each from a low melt gel. 
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Set up ligations 1.vector: pIG89 (XhoI/XbaI) + Insert: Qrr4/LuxO/U/CqsS (XhoI/XbaI) 2. pIG89 vector only control 3. vector: pIG10 (Hind/Xba) + insert: LuxO/U/CqsS (Hind/Xba) 4. pIG10 vector only control. Transformed. Three colonies from #1 and six colonies from #3 grew up.
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Set up overnight cultures of ligations 1 and 3 from 8/9. Three colonies from each ligation was used to innoculate 5ml of LB-AM. The next day did mini preps from each overnight and also froze down samples in the -80. NEED TO ENTER THESE STRAINS IN TO OUR DATABASE!!
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Even if we do manage to transform pIG87 into a cell that is plaque-susceptible, we don’t know if overexpressing CRO will cause lysis.  For that reason, Dr. Grose thinks it is wise for us to prepare a plasmid that overexpresses a gene known to cause lysis.  We know the E.Coli SOS pathway triggers phage lysis, so we are going to clone the RecA protein in the same position CRO is in, and then overexpress that protein … supposing that we figure out HOW to get a plasmid into the cell.  If we can’t do that, cloning RecA is a moot point.  We started the PCR reaction today.
Even if we do manage to transform pIG87 into a cell that is plaque-susceptible, we don’t know if overexpressing CRO will cause lysis.  For that reason, Dr. Grose thinks it is wise for us to prepare a plasmid that overexpresses a gene known to cause lysis.  We know the E.Coli SOS pathway triggers phage lysis, so we are going to clone the RecA protein in the same position CRO is in, and then overexpress that protein … supposing that we figure out HOW to get a plasmid into the cell.  If we can’t do that, cloning RecA is a moot point.  We started the PCR reaction today.
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Revision as of 05:54, 23 September 2013


Cholera Detection July-August Notebook: August 5 - August 18 Daily Log



Cholera Detection
March-April
May-June
July-August
September-October

8/7/2013

Using these strains that we know contain lytic phage, we’ve transformed pIG87 (CRO behind an arabinose-inducible promoter in a pLAT vector backbone) into 9901 and 9907 through the electroporation technique …

We plated them on arabinose, overexpressing CRO, but saw no plaques!! But even more mysteriously, when we perform a top agar H202 plaque assay on these post-electroporation strains, we see no plaques! Is lambda gone? Does the CRO protein actually inhibit, rather than initiate, lambda’s lytic cycle? Or does an excess of arabinose inhibit lysis?

KK, KP

Mini preped pIG78 again to have on hand in frozen stock. Froze down pIG12+Cro(PstI/EcoRI) as pIG87. This plasmid has been sequence verified.

Set up 30ul digests of pIG89 (XhoI, XbaI, NEB2), pIG78 (XhoI, XbaI, NEB2), pIG78 (HindIII, XbaI, NEB4), and pIG10 (HindIII, XbaI, NEB4). The first and second reactions' bands didn't separate well because they were so close in size. Redid these two in a triple digest adding BamHI. This helped separate out the bands.

Set up PCR for Bio Brick cloning for Cro (BI264/265) and for Qrr4/RFP (BI266/267)using phusion.

CH


8/8/2013

Set up 40ul Digests: 1. pIG89, XhoI, XbaI, NEB4 2. pIG78, XhoI, XbaI, BamHI, NEB4. 3. pIG78, HindIII, XbaI, BamHI, NEB4. 4. pIG10, HindIII, XbaI, NEB4. All the digests worked great. The addition of BamHI helped resolve the problem of the bands being to close together. Collected a slice of each from a low melt gel.

CH


8/8/2013

Set up 40ul Digests: 1. pIG89, XhoI, XbaI, NEB4 2. pIG78, XhoI, XbaI, BamHI, NEB4. 3. pIG78, HindIII, XbaI, BamHI, NEB4. 4. pIG10, HindIII, XbaI, NEB4. All the digests worked great. The addition of BamHI helped resolve the problem of the bands being to close together. Collected a slice of each from a low melt gel.

CH


8/9/13

Set up ligations 1.vector: pIG89 (XhoI/XbaI) + Insert: Qrr4/LuxO/U/CqsS (XhoI/XbaI) 2. pIG89 vector only control 3. vector: pIG10 (Hind/Xba) + insert: LuxO/U/CqsS (Hind/Xba) 4. pIG10 vector only control. Transformed. Three colonies from #1 and six colonies from #3 grew up.

CH


8/14/13
Top agar assay demonstrates our frozen TT9907 strain does indeed contain lambda prophage that can in turn indeed by excited to lyse the its host cell. No surprises there – that’s what we went through our selection process for in the first place (see July 31st and August 2nd entries). Frozen TT9901 failed to exhibit plaques. That is a surprise. Dr. Grose recommends we set the TT9901 aside for a moment and just use TT9907 to troubleshoot.

One explanation for our last experiment could be that something in the electroporation procedure itself inhibits downstream vulnerability to lambda infection. Perhaps the electric shock excises the prophage from the genome, or perhaps we select for E.Coli that is particularly invulnerable to lambda, or something.

Our electroporation protocol is as follows:

1) Centrifuge 1-2 mL overnight culture. Discard supernatant. 2) Glycerol Wash: resuspend cells in 500 uL 10% glycerol. Centrifuge 1 minute. Discard supernatant. 3) Resuspend cells in 500 uL of glycerol, and place on ice. Pipet 50 uL of cells into a microcentrifuge tube with 5 uL of plasmid. Pipet the solution into a silver-plated cuvette that has been sitting on ice. 4) Insert the cuvette into the electroporator and shock. 5) Return cells to ice for a few seconds. Draw up cells with 1 mL LB and pipet into a clean 2mL tube. 6) Incubate cells at 37°C for 30 minutes. 7) Plate 100-200 uL cells

To determine if the procedure itself interferes with lambda, we are going to take samples at each stage of the procedure – after resuspending in glycerol, after shocking, and after incubating – and do a H202 top agar assay to see compare plaques.

KK, KP

Set up overnight cultures of ligations 1 and 3 from 8/9. Three colonies from each ligation was used to innoculate 5ml of LB-AM. The next day did mini preps from each overnight and also froze down samples in the -80. NEED TO ENTER THESE STRAINS IN TO OUR DATABASE!!

CH


8/16/13
Shocking the cells significantly reduces the number of plaques we see compared to pre-shock cells suspended in glycerol! See the photo comparison of top agar plates before and after shocking. I give that an ambivalent “great.” It’s great that we’ve learned that bit of information; not-so-great that, well, how are we going to get a plasmid in there? We’re going to redo the experiment to confirm our results.

Even if we do manage to transform pIG87 into a cell that is plaque-susceptible, we don’t know if overexpressing CRO will cause lysis. For that reason, Dr. Grose thinks it is wise for us to prepare a plasmid that overexpresses a gene known to cause lysis. We know the E.Coli SOS pathway triggers phage lysis, so we are going to clone the RecA protein in the same position CRO is in, and then overexpress that protein … supposing that we figure out HOW to get a plasmid into the cell. If we can’t do that, cloning RecA is a moot point. We started the PCR reaction today.

KK, KP,CH


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