Team:EPF-Lausanne/Notebook/June2011

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Perhaps the ITT doesn't yield a functional TetR, or we didn't have the optimal concentration of binding DNA (too much nonspecific interactions?). We'll definitely continue the MITOMI practise to find out.
Perhaps the ITT doesn't yield a functional TetR, or we didn't have the optimal concentration of binding DNA (too much nonspecific interactions?). We'll definitely continue the MITOMI practise to find out.
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== Tuesday, June 28 2011 ==
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In order to avoid putting the lysis cassette and the LacI-TetR construct all on the same plasmid, the assembly designers chose to use a two-plasmid strategy. To get things started, we took the P23019 plasmid and the J61002 plasmid from the iGEM registry. The p23019 plasmid comes with chloramphenicol resistance, while the J61002 plasmid comes with ampicillin resistance and an RFP (red fluorescent protein) gene.
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Henrike and Irina designed primers for both plasmids, with the goal of amplifying the following four parts:
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1) a pTet-RFP segment (820 bp)
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*Primer 1: J61002-Ptet-RFP-r
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*Primer 2: J61002-RFP-f
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*DNA template: Plate 1, 18 C
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2) a J61002 plasmid backbone with a terminator (2334 bp)
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*Primer 1: J61002-f
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*Primer 2: J61002-term-r
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*DNA template: Plate 1, 18 C
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3) a constitutive promoter for TetR (725 bp)
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*Primer 1: Pconst-tetR-f
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*Primer 2: TetR-r
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*DNA template: TetR Repressilator plasmid
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4) P23019 plasmid (2242 bp)
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*Primer 1: P23019-r
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*Primer 2: P23019-f
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*DNA template: Plate 4, 14E
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With a first PCR, we were able to amplify the pTet-RFP segment and the promoter for TetR, but neither of the backbones was amplified. The iProof PCR was done using 40 seconds of elongation time at 55 C.
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[[File:EPFL-28-06-pcr.jpg‎|300px|TetR segment and RFP-pTet segment amplified. Both backbones unamplified]]
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== Wednesday, 29 June 2011 ==
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The mixed success of the previous day's PCR required a change in tactics. First, it was decided that the elongation time should be increased from 40 seconds to 1 minute. Second, it was deemed important to take melting temperature into consideration. The IDT (Integrated DNA Technologies) toolbox allows you to compute melting temperatures for sequences. Inserting the primer sequences yielded the following heats:
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1) P23019-r: 45.8 + 3 = 48.8
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2) P23019-f: 60.8 + 3 = 63.8
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3) J61002-bb-f: 52.2 + 3 = 55.2
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4) J61002-term-r: 49.3 + 3 = 52.3
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The lowest temperature was 49 (48.8) so we proceeded to do a PCR with these values. The J61002 plasmid backbone was properly amplified.However, the P23019 backbone remained unamplified despite these changes.
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[[File:EPFL-29-06_J6_backb.jpg|300px|J61002 backbone amplification, but no P23019]]
== Thursday, 30 June 2011 ==
== Thursday, 30 June 2011 ==
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== Gibson Assembly==
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== Friday, 1st of July 2011==
 
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Alina and Lilia did MITOMI on His-tetR expressed from linear template, it was loaded on chip in ITT expression mix. DNA was spotted on June 29 in different concentrations for both: consensus (tetO1) sequence and a random sequence ((-)control).
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On the Gibson assembly side of things, Vincent and Irina purified the PCR product of both the J61002 plasmid and the pTet-RFP construct using the standard PCR purification protocol. Then, using NanoDrop, they measured the concentrations of J61002 plasmid and of the RFP construct:
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[[File:EPFL_1jully_MITOMI_His-TetR_concensus_vs_negctrl.png|600px|GFP-tetR and cy5 fluorescence‎]]
 
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Images: Green fluorescence comes from green-lysine and red fluorescence comes from DNA (cy-5 labeled).  
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RFP (820 bp) : 28.8 ng/microL
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Although we don't observe much protein bound to the anti His-tag antibody, we can still see that it does bound TetO1 sequence and did not bind any (-)control sequence. 
 
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Some possible reasons for low protein fluorescence under the buttons: the expression yield from the linear template with T7 promoter might be too low, the His-tag to antibody binding is not strong enough or the concentration of antibody is too low.
 
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J6 (2334 bp) : 26 ng/microL
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== Friday, 1st of July 2011==
 
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Alina and Lilia amplified the His-tetR linear template. After PCR sample was loaded on 1% agarose gel with Rad Safe, “1Kb Plus DNA Ladder” was used.
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'''Instead of following the instructions on the wiki by Nikolaus Obholzer which has the wrong formula for finding the equal molecular ratio''', we determined that the ratio ought to be 80/233 since the length of the RFP fragment is 820 bp and the length of the J6 plasmid is 2334. To make the Gibson mix, we used 2.6 microliters (80 ng % 28.8 ng/microL) of the RFP segment and 9 microliters of the plasmid (230 ng % 26 ng/microL). The assembly was done with a sample of 5 microliters taken from the 11.6 microliter mix.  
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We then did a transformation with these Gibson-ed plasmids and plated the cells on ampicillin plates and put them in the incubator overnight.
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==  Gradient PCR  ==
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In a last attempt to try to amplify the P23019 plasmid backbone (the last piece needed to start a Gibson assembly of the TetR plasmid), Vincent tried a gradient PCR in which separate samples of the plasmid would be run at temperatures between 40 and 50 C (40, 42, 44, 46, and 48). The results showed, once again, that amplification was beyond reach.
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[[File:EPFL-29-06-p23019fail.jpg|300px|GFP-tetR and cy5 fluorescence]]
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Latest revision as of 16:51, 9 July 2011

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