Team:EPF-Lausanne/Notebook/July2011

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[[File:EPFL-08-07_igem_tetRvariants_1.jpg|thumb|right|Gel from the tetR mutants PCR. 1000kb ladder.]]
[[File:EPFL-08-07_igem_tetRvariants_1.jpg|thumb|right|Gel from the tetR mutants PCR. 1000kb ladder.]]
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We ran the PCR on the tetR linear template, using Clara's primers for site-specific mutagenesis.
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We ran the PCR on the tetR linear template, using Clara's primers for PCR mutagenesis.
Six PCRs were run. The first amplified the common sequence of the mutants: everything up to the mutated sites. The six other reactions amplified the second half of the gene, inducing specific mutations in tetR.
Six PCRs were run. The first amplified the common sequence of the mutants: everything up to the mutated sites. The six other reactions amplified the second half of the gene, inducing specific mutations in tetR.
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# YF42
# YF42
# PQ39YM42
# PQ39YM42
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# PQ39LV41M42
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# PQ39LV41YM42
The gel fluorescence traces show what appears to be PCR product in large amounts for the Common and EA37 reactions, and smaller amounts for the YF36 and YF42 reactions. The remaining three show almost no activity.
The gel fluorescence traces show what appears to be PCR product in large amounts for the Common and EA37 reactions, and smaller amounts for the YF36 and YF42 reactions. The remaining three show almost no activity.
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# Y42F
# Y42F
# P39Q_Y42M
# P39Q_Y42M
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# P39Q_L41V_Y42M
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# P39Q_L41V ''([26 July 2011] The Y42M mutation was left out here)''
<s>By laziness</s> For convenience, the primer codes were changed to match those used by Agilent's primer design software. For example, V36F indicates a substitution of amino acid F for V in position 36. The primers are listed in this Google spreadsheet: https://spreadsheets.google.com/spreadsheet/ccc?key=0AvMKvyibLKKxdENDWkNLRV9ZSHhvVU45RHQ0bnNCWFE&hl=en_US
<s>By laziness</s> For convenience, the primer codes were changed to match those used by Agilent's primer design software. For example, V36F indicates a substitution of amino acid F for V in position 36. The primers are listed in this Google spreadsheet: https://spreadsheets.google.com/spreadsheet/ccc?key=0AvMKvyibLKKxdENDWkNLRV9ZSHhvVU45RHQ0bnNCWFE&hl=en_US
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Douglas prepared agar-ampicillin plates for the mutagenesis, and transformed a pF3A plasmid with GFP (and ampicillin resistance). It can be used for a trial run of mutagenesis if the gibson assembled tetR plasmid is not yet available. He also transformed some repressilator, so we should have plenty to keep us going for a while. Also, Nadine debriefed him on her work for the past few weeks, so he can take over while she is on holiday.
Douglas prepared agar-ampicillin plates for the mutagenesis, and transformed a pF3A plasmid with GFP (and ampicillin resistance). It can be used for a trial run of mutagenesis if the gibson assembled tetR plasmid is not yet available. He also transformed some repressilator, so we should have plenty to keep us going for a while. Also, Nadine debriefed him on her work for the past few weeks, so he can take over while she is on holiday.
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== Thursday, 21 July 2011 ==
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Douglas and Alessandro inoculated the colonies transformed with the repressilator plasmid to make minipreps the following day. Alessandro also made the LB broth (because one bottle got contaminated) and distilled water.
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[[File:EPFL-2011-07-21_Gibson_detector_fragments_PCR.jpg|thumb|right|Gel from our first attempt to replicate Nadine's PCR, to create fragments for the J61002-based reporter plasmid. None of the expected products show up: they should all be between 700 b and 2.4 kb]]
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Douglas and Alessandro repeated Nadine's unsuccessful PCR, to make the '''fragments for Gibson assembly of the 'reporter plasmid' ''' (pTet-lacI-pLac-RFP/Lysis). The PCR was just as '''unsuccessful''', showing no trace of the expected products, as shown on the figure to the right. The fragments being assembled are:
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* A J61002 '''backbone''' amplified from the J61002-pTet-RFP plasmid
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* '''LacI''' amplified from the Repressilator plasmid
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* '''pLac-RFP''' from the T4 Lysis plasmid
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* OR '''pLac-T4Lysis''' also from the J61002-pTet-RFP plasmid
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We found two similarly labelled tubes containing J61002-pTet-RFP, so we used both for our PCRs. The 'bis' reactions used the plasmid miniprepped by Allesandro, labelled in green writing. The 'non-bis' reactions used the other plasmid, of mysterious origin (miniprepped by Vincent perhaps?).
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New reagents were prepared to repeat the PCR, using this time the optimal concentrations recommended by Bio-Rad for the iProof polymerase, i.e. primer concentrations of 0.5 uM and DNA template quantities between 1 pg and 10 ng for 50 ul. Specifically, we prepared a set with 1:1000 diluted template (resulting in a mass between 25 and 100 pg) and a set with 1:100 diluted template (resulting in a mass between 0.25 and 1 ng). They have been frozen with no polymerase, to attempt a touchdown PCR tomorrow.
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== Friday, 22 July 2011 ==
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Alessandro made minipreps for the repressilator plasmid.
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[[File:EPFL-2011-07-22_Gibson_TD_PCR_reporter.jpg|thumb|right|*EPIC FAIL* - Touchdown PCR to amplify the fragments for Gibson assembly of the two reporter plasmids]]
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'''Touchdown PCR''' was run on yesterday's samples, to amplify the '''fragments for Gibson assembly of the reporter plasmids''', using the Bio-Rad thermal cycler. The thermal cycles were designed according to the iProof datasheet, using touchdown PCR recommendations from Korbie & Mattick (2008) [1]. Since the primer melting temperatures range from 47° C to 60° C, the cycle step the annealing temperature down from 65° C to 45° C over 20 cycles in constant decrements. It then repeats another 20 cycles at the 45° C floor temperature. Elongation lasts 40 s, following Bio-Rad's recommendation of 15 s/kb, for a 2400 kb product. The other parameters are as usual for iProof polymerase.
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The PCR yielded absolutely '''no products'''. To further investigate the cause of failure, the PCR has been '''repeated''' using the Roche '''High Fidelity PLUS''' PCR kit, adapting the thermal cycles to the new polymerase. In a desparate move, the old PCR products were re-used for an almost-identical run on the Eppendorf cycler, with slightly longer elongation and denaturation steps (50 s and 7 s, respectively).
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1. Korbie, D.J. & Mattick, J.S. Touchdown PCR for increased specificity and sensitivity in PCR amplification. Nature protocols 3, 1452-6(2008).
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== Monday, 25 July 2011 ==
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[[File:EPFL-2011-07-25_Gibson_TD_PCR_HifiPLUS.jpg|thumb|right|Gibson fragments for the reporter plasmid, PCR'd with HifiPLUS enzyme. Good yield, but some unexpected product sizes, especially for the backbone.]]
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Alessandro ran the Gel on Friday's PCR - that made Gibson fragments for the reporter plasmid, using the HifiPLUS enzyme. There is definitely some product (whereas iProof yields nothing at all). LacI and RFP seem to have worked properly. The lysis fragment is possibly a little short, and the backbone band is way too small. Overall, except for the backbone, it worked well.
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In conclusion: our '''iProof enzyme seems not to work ''', either because it degraded, or it is just not suitable for this type of experiment. We should now use another enzyme. We ordered some High Fidelity PLUS for the next PCRs.
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To investigate the lack of backbone product, Douglas ran a gradient PCR for the backbone fragment and the T4Lysis fragment. Five annealing temperatures were tested for each. The row temperatures are listed in the table below. To straddle their primers' expected annealing temperature, the backbone was placed in rows D--H and the Lysis fragment in rows A--E. Please note this PCR was run with the '''standard High Fidelity (not PLUS)''' enzyme!
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{|
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|+ Temperature settings for the gradient PCR
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! Row || Temperature [°C]
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|-
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| A || 65.0
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|-
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| B || 63.8
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|-
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| C || 61.6
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|-
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| D || 57.6
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|-
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| E || 52.7
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|-
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| F || 48.9
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|-
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| G || 46.2
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|-
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| H || 45.0
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|}
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== Tuesday, 26 July 2011 ==
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Alessandro made PCR to amplify pSB3K1 backbone to use for a gibson assembly to make the Pconst-TetR plasmid. We can't do the assembly now since we are waiting for primers for TetR.
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The PCR gave good result for one colony (on two) as we can see a big band of the expected size (3.1 kb).
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This time the purification will be performed not from the gel but directly from PCR with a commercial kit.
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[[File:EPFL2011_pSB3K1_PCR_260711.jpg|thumb|right|pSB3K1 successfully amplified by PCR]]
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Alessandro send the Vincent's plasmid (J6001-Ptet-RFP) to be sequenced (as the primer arrived).
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----------
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All the mutagenesis primers have been delivered, so Douglas diluted them. They are now all at 1 ug/ul concentrations, and need a further 1:10 or sodilution before being used for mutagenesis. He then ran a first mutagenesis on Alina's pF3A-tetR-GFP plasmid, introducing the following five mutations, in addition to a control reaction:
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# V36F
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# E37A
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# P39K
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# P39Q_Y42M
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# P39Q_L41V
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The mutagenesis kit (remains from last year) was almost empty, so he made 10 ul instead of 50 ul reactions. There should be enough remaining reagents for one more batch, possibly with control, but hardly any more.
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The mutated plasmids were frozen over night, in order to digest and transform the following day.
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A gel was run for the gradient PCR, that used HiFi enzyme. It gave no product! In conclusion, we shall wait for the High Fidelity PLUS Enzyme, which should get here tomorrow.
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In the mean time, Doug ran an identical gradient PCR with Matt's HiFiPLUS enzyme, changing the buffer concentration to 10 ul per 50 ul and lengthening the extension step to 2'30", as recommended by the datasheet.
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== Wednesday, 27 July 2011 ==
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Alessandro purified the pSB3K1 backbone (to use for Gibson assembly and make the Pconst-TetR plasmid) from the PCR of the day before getting 60.6 ng/uL.
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The sequencing reaction failed and checking the conditions of primers and template with NanoDrop, it seems like reverse primer has no DNA (but this doesn't explain why the reaction failed also with the forward primer); there's also a weird thing to annotate: checking the template on NanoDrop we always have an error of calibration.
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Alessandro made new sample to send again sequencing.
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---------------
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We '''FINALLY HAVE A WORKING PCR!!!!1111one''''.
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[[File:EPFL-2011-07-27_Gibson_gradient_PCR_hifiPLUS.jpg|thumb|right|Backbone and Lysis fragments for the reporter plasmids, PCR'd with HifiPLUS. Expected product lengths: 2.4 kb backbone, 1.9 kb Lysis cassette.]]
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More seriously, a gel was run on yesterday's gradient PCR products --- the PCR was ran using the High Fidelity PLUS enzyme, on the J61002-pTet backbone and pLac-T4Lysis fragments, for the Gibson assembly of the reporter plasmids. For both fragments, high quantities of specific product were yielded, though the backbone also contains large amounts of non-specific product.
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For the backbone, lower annealing temperatures yield more product, as expected. The non-specific product seems relatively unaffected by annealing temperature (or it may decrease slightly at lower temperatures). The unspecific product present in large amounts is of similar length to the RFP gene, which is also present on the backbone template --- but determining its origin is unnecessary: it doesn't contain an antibiotic resistance gene, so plasmids assembled from it would not allow culture growth in the amplification step.
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The T4 Lysis cassette fragment is a little too small. It could be due to GelRed affecting migration speed (according to Alina, it happens with large amounts of DNA), or it could indeed be an undesired product of shorter length. Please note the product quantities are barely affected by temperature (to the extent we can eyeball from the gel), whereas the expected primer melting temperatures are between 52° and 57°C, so we do not expect any product with 65°C annealing steps.
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The assembly step was started. Transformation can be started tomorrow morning.
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'''For the following PCRs''': Use Roche High Fidelity PLUS enzyme, and adapt any protocols to the appropriate specifications by Roche. Also, use either bottled water or the newer jar with fresher water (the one with autoclave tape on the lid).
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== Thursday, 28 July 2011 ==
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The sequencing reaction this time went good therefore the plasmid is definitely the one expected.
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Alessandro transformed and plated the plasmid to perform the day after the colony PCR just to optimize the protocol (this will be our positive control) for this technique that will be used to screen the gibson assembly.
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Since seems to be some debris in the SOC medium bottle, we also plated it (under the same conditions of the cells) to test if something grows. Alessandro also tested the OD of the medium after 1 hour of incubation and no significant difference was found.
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------
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Douglas transformed and plated the Gibson-assembled reporter plasmids at the same time as Alessandro's plasmids. He later transformed it in a different ''E. Coli'' colony that over-expressed LacI, and therefore has a reduced probability of expressing the holin (from the lysis cassette) before they synthesise enough LacI to repress it.
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Douglas also repeated the '''mutation PCR''' using the new High Fidelity PLUS enzymes. In a bold and dareful move, he programmed the thermal cycler to carry out both a touchdown PCR AND progressively extended elongation steps (during the second set of cycles, those at the floor annealing temperature, the elongation step is extended of five seconds every cycle).
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== Friday, 29 July 2011 ==
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Douglas and Alessandro's transformed cells didn't grow (maybe problems with competent cells?).
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Alessandro transformed the cells again (with the J61002-Ptet-RFP Gibson plasmid) but this time he trasformed also the sample labeled Col1-RFP and Col4-RFP (supposing that these are the same as J61002-Ptet-RFP Gibson plasmid).
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Alessandro transformed the Gibson assembly made by Douglas on Lilia's (or Alina's?) competent cells and also on Henrike's competent cells. Due to an error only the lysis cassette has been transformed. On Tuesday the Gibson assembly has to be repeated since we ran out of the previous one.
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The Mutation PCR yielded no product, probably because the primers were too diluted. It has been repeated with a higher primer concentration.
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Latest revision as of 16:18, 2 August 2011

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