Team:EPF-Lausanne/Notebook/June2011

From 2011.igem.org

(Difference between revisions)
(→Gibson Assembly)
 
(57 intermediate revisions not shown)
Line 1: Line 1:
{{:Team:EPF-Lausanne/Templates/Header|title=Notebook: June 2011}}
{{:Team:EPF-Lausanne/Templates/Header|title=Notebook: June 2011}}
-
== 3 June 2011 ==
+
== Friday, 3 June 2011 ==
Lysis device sequences were received from Microsynth. Only two reactions worked: those containing primers 1289F and 1333R.
Lysis device sequences were received from Microsynth. Only two reactions worked: those containing primers 1289F and 1333R.
Line 49: Line 49:
   NNNNN
   NNNNN
 +
== Monday, 6 June 2011 ==
 +
 +
Irina and Nadine selected the 2 plasmid backbones we will use: J23019 and J61002 (check the parts registry). They have 2 different origin of replication sites and should both have 15-20 copy numbers. Interesting also, they both contain RFP and pTet. We already have them in the freezers, now let's focus on primers design!
 +
 +
 +
== Wednesday, 8 June 2011 ==
 +
 +
Lilia and Douglas miniprep'ed the cultures containing the lysis cassette, and sent the plasmids for sequencing.
 +
 +
Three colonies were miniprep'ed: two containing the T4 variant, one containing the lambda variant. This yielded solutions with plasmid concentrations of 221.9 ng/µl for T4 colony 1, 225.5 ng/µl for T4 colony 2, and 57.0 ng/µl for lambda colony.  Only T4 colony 1 was sent for sequencing, using the primer solutions prepared on [[Team:EPF-Lausanne/Notebook/May2011|31 May 2011]]. Results should be in on Friday.
 +
 +
== Friday, 10 June 2011 ==
 +
 +
Sequencing results from Microsynth for the lysis cassette (Biobrick K112808) have arrived. The sequence is entirely covered by the seven reactions, and all but a few bases match. As far as I can tell, this biobrick is valid! (Doug)
 +
 +
The results of a blast, individually querying each of the sequences against the official K112808 sequence are available here:  [https://static.igem.org/mediawiki/2011/7/73/EPFL-2011-06-10-Lysis_BLAST.txt BLAST results].
 +
 +
Sequencing results, for each primer (again, reverse complements shown here for the reverse primers --- those denoted by an "R"):
 +
 +
    >1558332 30F
 +
    NGNNNTCATTTGGTGTTCAGATCGCTTGTTCAAGATAACGCTACCGGGAAGGTTCTTGCTTCCCGGGTAGCTGTCGTAATTCTTTTGTTTATAATGGCGA
 +
    TTGTTTGGTATAGGGGAGATAGTTTCTTTGAGTACTATAAGCAATCAAAGTATGAAACATACAGTGAAATTATTGAAAAGGAAAGAACTGCACGCTTTGA
 +
    ATCTGTCGCCCTGGAACAACTCCAGATAGTTCATATATCATCTGAGGCAGACTTTAGTGCGGTGTATTCTTTCCGCCCTAAAAACTTAAACTATTTTGTT
 +
    GATATTATAGCATACGAAGGAAAATTACCTTCAACAATAAGTGAAAAATCACTTGGAGGATATCCTGTTGATAAAACTATGGATGAATATACAGTTCATT
 +
    TAAATGGACGTCATTATTATTCCAACTCAAAATTTGCTTTTTTACCAACTAAAAAGCCTACTCCCGAAATAAACTACATGTACAGTTGTCCATATTTTAA
 +
    TTTGGATAATATCTATGCTGGAACGATAACCATGTACTGGTATAGAAATGATCATATAAGTAATGACCGCCTTGAATCAATATGTGCTCAGGCGGCCAGA
 +
    ATATTAGGAAGGGCTAAATAATTAACTAGAATACTTAGGAGGTATTATGAATATATTTGAAATGTTACGTATAGATGAAGGTCTTAGACTTAAAATCTAT
 +
    AAAGACACAGAAGGCTATTACACTATTGGCATCGGTCATTTGCTTACAAAAAGTCCATCACTTAATGCTGCTAAATCTGAATTAGATAAAGCTATTGGGC
 +
    GTAATTGCAATGGTGTAATTACAAAAGATGAGGCTGAAAAACTCTTTAATCAGGATGTTGATGCTGCTGTTCGCGGAATCCTGAGAAATGCTAAATTAAA
 +
    ACCGGTTTATGATTCTCTTGATGCGGTTCGTCGCTGTGCATTGATTAATATGGTTTTCCAAATGGGANAAACCGGTGTGGCAGGATTTACTAACTCTTTA
 +
    CGTATGCTTCAACAAAAACGCTGGGATGAAGCAGCAGTTAACTTAGCTAANAGTAGATGGNNTAATCAAACNCCTAATCNNNNAAAACGAGTCATTACAA
 +
    CGTTTANAACTGGNNN
 +
   
 +
    >1558333 440F
 +
    NNNATTCCNACTCAAATTTGCTTTTTTACCAACTAAAAAGCCTACTCCCGAAATAAACTACATGTACAGTTGTCCATATTTTAATTTGGATAATATCTAT
 +
    GCTGGAACGATAACCATGTACTGGTATAGAAATGATCATATAAGTAATGACCGCCTTGAATCAATATGTGCTCAGGCGGCCAGAATATTAGGAAGGGCTA
 +
    AATAATTAACTAGAATACTTAGGAGGTATTATGAATATATTTGAAATGTTACGTATAGATGAAGGTCTTAGACTTAAAATCTATAAAGACACAGAAGGCT
 +
    ATTACACTATTGGCATCGGTCATTTGCTTACAAAAAGTCCATCACTTAATGCTGCTAAATCTGAATTAGATAAAGCTATTGGGCGTAATTGCAATGGTGT
 +
    AATTACAAAAGATGAGGCTGAAAAACTCTTTAATCAGGATGTTGATGCTGCTGTTCGCGGAATCCTGAGAAATGCTAAATTAAAACCGGTTTATGATTCT
 +
    CTTGATGCGGTTCGTCGCTGTGCATTGATTAATATGGTTTTCCAAATGGGAGAAACCGGTGTGGCAGGATTTACTAACTCTTTACGTATGCTTCAACAAA
 +
    AACGCTGGGATGAAGCAGCAGTTAACTTAGCTAAAAGTAGATGGTATAATCAAACACCTAATCGCGCAAAACGAGTCATTACAACGTTTAGAACTGGCAC
 +
    TTGGGACGCGTATAANAATCTATAAAGCACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTG
 +
    TCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATACTAGAGTTGACAGCTAGCTCAGTCCTAGGGACTA
 +
    TGCTAGCTACTAGAGATAGGAGGCCTTTATGGCCTTAAAAGCAACAGCACTTTTTGCCATGCTAGGATTGTCATTTGTTTTATCTCCATCGATTGAAGCG
 +
    AATGTCNATCCTCATTTTGATAAATTTATGGAATCTGGNNTTAGGCACGTTTATNTGCTTTTTGAAAANAAAANCGTANAATCNNCTGAACAATTCTATA
 +
    GTTTTANNNNAACGACCTATAAAANTGACCCNNGN
 +
   
 +
    >1558334 947F
 +
    NNTTGNNGCGGTTCGTCGCTGTGCATTGATTAATATGGTTTTCCAAATGGGAGAAACCGGTGTGGCAGGATTTACTAACTCTTTACGTATGCTTCAACAA
 +
    AAACGCTGGGATGAAGCAGCAGTTAACTTAGCTAAAAGTAGATGGTATAATCAAACACCTAATCGCGCAAAACGAGTCATTACAACGTTTAGAACTGGCA
 +
    CTTGGGACGCGTATAAAAATCTATAAAGCACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTT
 +
    GTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATACTAGAGTTGACAGCTAGCTCAGTCCTAGGGACT
 +
    ATGCTAGCTACTAGAGATAGGAGGCCTTTATGGCCTTAAAAGCAACAGCACTTTTTGCCATGCTAGGATTGTCATTTGTTTTATCTCCATCGATTGAAGC
 +
    GAATGTCGATCCTCATTTTGATAAATTTATGGAATCTGGTATTAGGCACGTTTATATGCTTTTTGAAAATAAAAGCGTAGAATCGTCTGAACAATTCTAT
 +
    AGTTTTATGAGAACGACCTATAAAAATGACCCGTGCTCTTCTGATTTTGAATGTATAGAGCGAGGCGCGGAGATGGCACAATCATACGCTAGAATTATGA
 +
    ACATTAAATTGGAGACTGAATGANATACTAGAGCCGGCTTATCGGTCAGTTTCACCTGATTTACGTAAAAACCCGCTTCGGCGGGTTTTTGCTTTTGGAG
 +
    GGGCAGAAAGATGAATGACTGTCCACGACGCTATACCCAAAAGAAATACTAGTCTGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCT
 +
    GCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAG
 +
    GCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCNCAGGCTCCGCCCCCCTGACGANCATCNCAAAAATCGACGCTCAAGTCANANGNN
 +
   
 +
    >1558335 1289F
 +
    NNCTTCGGNGGGCTTTCTGNGTTTATATANTAGAGTTGACAGCTAGCTCAGTCCTAGGGACTATGCTAGCTACTAGAGATAGGAGGCCTTTATGGCCTTA
 +
    AAAGCAACAGCACTTTTTGCCATGCTAGGATTGTCATTTGTTTTATCTCCATCGATTGAAGCGAATGTCGATCCTCATTTTGATAAATTTATGGAATCTG
 +
    GTATTAGGCACGTTTATATGCTTTTTGAAAATAAAAGCGTAGAATCGTCTGAACAATTCTATAGTTTTATGAGAACGACCTATAAAAATGACCCGTGCTC
 +
    TTCTGATTTTGAATGTATAGAGCGAGGCGCGGAGATGGCACAATCATACGCTAGAATTATGAACATTAAATTGGAGACTGAATGAAATACTAGAGCCGGC
 +
    TTATCGGTCAGTTTCACCTGATTTACGTAAAAACCCGCTTCGGCGGGTTTTTGCTTTTGGAGGGGCAGAAAGATGAATGACTGTCCACGACGCTATACCC
 +
    AAAAGAAATACTAGTCTGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATAC
 +
    GGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTT
 +
    CCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCT
 +
    GGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCAC
 +
    GCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTA
 +
    TCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAANAGGATTAGCANNNNNNGGTATGTAGGNNNNNCTACAGAN
 +
    TTCTTGAAGTGGNGGCCTAACTACGNNTAN
 +
   
 +
    >1558336 816R
 +
    NTTCAGNNTNTTTTACTTTCACCAGCGTTTNTGGGNGANNAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGNGACACGGAAATGTTGAAT
 +
    ACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTNTCAGGGTTATTGTNTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATA
 +
    GNGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGA
 +
    ATNTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGACTTAAAAGGAGGGTCTATGGCAGCACCTAGAAT
 +
    ATCATNTTCGCCCTCTGATATTCTATTTGGTGTTCTAGATCGCTTGTTCAAAGATAACGCTACCGGGAAGGTTCTTGCTTCCCGGGTAGCTGTCGTAATT
 +
    CTTTTGTTTATAATGGCGATTGTTTGGTATAGGGGAGATAGTTTCTTTGAGTACTATAAGCAATCAAAGTATGAAACATACAGTGAAATTATTGAAAAGG
 +
    AAAGAACTGCACGCTTTGAATCTGTCGCCCTGGAACAACTCCAGATAGTTCATATATCATCTGAGGCAGACTTTAGTGCGGTGTATTCTTTCCGCCCTAA
 +
    AAACTTAAACTATTTTGTTGATATTATAGCATACGAAGGAAAATTACCTTCAACAATAAGTGAAAAATCACTTGGAGGATATCCTGTTGATAAAACTATG
 +
    GATGAATATACAGTTCATTTAAATGGACGTCATTATTATTCCAACTCAAAATTTGCTTTTTTACCAACTAAAAAGCCTACTCCCGAAATAAACTACATGT
 +
    ACAGTTGTCCATATTTTAATTTGGATAATATCTATGCTGGAACGATAACCATGTACTGGTATAGAAATGATCATATAAGTAATGACCGCCTTGAATCAAT
 +
    ATGTGCTCAGGCGGCCAGAATATTAGGAAGGGCTAAATAATTAACTAGAATACTTAGGAGGTATTATGAATATATTTGAAATGTTACGTATAGATNAAGG
 +
    TCTAGACTAAANNCN
 +
   
 +
    >1558337 1333R
 +
    CCGGGAAGGTTNTTNNNTCCCGGGTAGNNNNCGTAATNNTTNNNTNNNAATGNNGATTGTTTGTNTAGGGGAGATAGTTTNTTTGAGTACNATAAGCAAT
 +
    CAAAGTNTGAAACATCCAGNGAAATTATTGAAAAGGAAANAACTGCACNCTTTGAATCTNTNGCCCTGGAACAACTCCAGATAGTTCATATNTCATNTGA
 +
    GGCAGACTTTAGTGCGGTGTATTCTNTCCGCCCTAAAAACTTAAACTATTTTGTTGATATTATAGCATACGAAGGAAAATTACCTTCAACAATAAGTGAA
 +
    AAATCACTTGGAGGATATCCTGTTGATAAAACTATGGATGAATATACAGTTCATTTAAATGGACGTCATTATTATTCCAACTCAAAATTTGCTTTTTTAC
 +
    CAACTAAAAAGCCTACTCCCGAAATAAACTACATGTACAGTTGTCCATATTTTAATTTGGATAATATCTATGCTGGAACGATAACCATGTACTGGTATAG
 +
    AAATGATCATATAAGTAATGACCGCCTTGAATCAATATGTGCTCAGGCGGCCAGAATATTAGGAAGGGCTAAATAATTAACTAGAATACTTAGGAGGTAT
 +
    TATGAATATATTTGAAATGTTACGTATAGATGAAGGTCTTAGACTTAAAATCTATAAAGACACAGAAGGCTATTACACTATTGGCATCGGTCATTTGCTT
 +
    ACAAAAAGTCCATCACTTAATGCTGCTAAATCTGAATTAGATAAAGCTATTGGGCGTAATTGCAATGGTGTAATTACAAAAGATGAGGCTGAAAAACTCT
 +
    TTAATCAGGATGTTGATGCTGCTGTTCGCGGAATCCTGAGAAATGCTAAATTAAAACCGGTTTATGATTCTCTTGATGCGGTTCGTCGCTGTGCATTGAT
 +
    TAATATGGTTTTCCAAATGGGAGAAACCGGTGTGGCAGGATTTACTAACTCTTTACGTATGCTTCAACAAAAACGCTGGGATGAAGCAGCAGTTAACTTA
 +
    GCTAAAAGTAGATGGTATAATCAAACACCTAATCGCGCAAAACGAGTCATTACAACGTTTAGAACTGGCACTTGGGACGCGTATAAAAATCTATAAAGCA
 +
    CTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCNTTCGNTTNN
 +
   
 +
    >1558338 1734R
 +
    TCCAACTCAAAATTTNNTTTNTTNCCAACTAAAAANNCTNNTCCCGAAATAAACTNNANGNNNAGTNNTCCANNTNTTAATTTGGATAANNNNTNNGCTG
 +
    GAACGATAACCATGTACTGGTATAGAAATGATCANNNAAGTAATGACCNCCTTGAATCAATATGTGNTCAGGNGNCCAGAATATTAGGAAGGGCTAAATA
 +
    ATTAACTAGAATACTTAGGAGGTATTATGAATATATTTGAAATGTTACGTATAGATGAAGGTNTTAGACTTAAAATCTATAAAGACACAGAAGGCTATTA
 +
    CACTATTGGCATCGGTCATTTGCTTACAAAAAGTCCATCACTTAATGCTGCTAAATCTGAATTAGATAAAGCTATTGGGCGTAATTGCAATGGTGTAATT
 +
    ACAAAAGATGAGGCTGAAAAACTCTTTAATCAGGATGTTGATGCTGCTGTTCGCGGAATCCTGAGAAATGCTAAATTAAAACCGGTTTATGATTCTCTTG
 +
    ATGCGGTTCGTCGCTGTGCATTGATTAATATGGTTTTCCAAATGGGAGAAACCGGTGTGGCAGGATTTACTAACTCTTTACGTATGCTTCAACAAAAACG
 +
    CTGGGATGAAGCAGCAGTTAACTTAGCTAAAAGTAGATGGTATAATCAAACACCTAATCGCGCAAAACGAGTCATTACAACGTTTAGAACTGGCACTTGG
 +
    GACGCGTATAAAAATCTATAAAGCACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGG
 +
    TGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATACTAGAGTTGACAGCTAGCTCAGTCCTAGGGACTATGCT
 +
    AGCTACTAGAGATAGGAGGCCTTTATGGCCTTAAAAGCAACAGCACTTTTTGCCATGCTAGGATTGTCATTTGTTTTATCTCCATCGATTGAAGCGAATG
 +
    TCGATCCTCATTTTGATAAATTTATGGAATCTGGTATTAGGCACGTTTATATGCTTTTTGAAAATAAAAGCGTAGAATCGTCTGAACAATTCTATAGTTT
 +
    TATGAGAACGACCTATAAAAATGACCCGTGCTCTTCTGATTTTGAATGTATAGAGCGAGGCGCGGAGANNNCACAN
 +
 +
 +
== Tuesday, 21 June 2011 ==
 +
 +
With help from Henrike, Douglas checked the mistakes in the K112808 (lysis cassette) sequence. On the entire sequence, only 7 errors occurred in several reactions, and are therefore likely to be actual errors in the DNA. They occur in positions (with regard to the original sequence in the registry):
 +
 +
* '''70''': CTC codon substituded for CTA. Both code for ''leu'', so that position is still valid
 +
 +
The following are missing bases:
 +
 +
* '''677''' and '''684''': lie in the junction between sub-bricks K112805 and K112806. Still valid.
 +
* '''1199''': lies in the junction between K112806 and B0010. Still valid.
 +
* '''1387''': second base of K112807, in the non-coding region. Still valid.
 +
* '''1698''': lies in the junction between K112807 and B0010. Still valid.
 +
* '''1701''': beginning of B0010, in the non-coding region. Still valid.
 +
 +
In conclusion, there is one codon substitution, and six missing bases that all lie within the junction between two bricks or the non-coding region of a brick. Experimentally, the brick does lyse cells in culture. Therefore, the sequence appears valid.
 +
 +
== Monday, 27 June 2011 ==
 +
 +
We practised MITOMI with TetR linear template and random DNA sequence + target DNA sequence. We performed ITT (in vitro transcription/translation) on TetR linear template before adding it to the chip. However, the results are disappointing: we have more DNA binding for the random sequence (1000 RFU change) than for the target sequence (500 RFU)...
 +
 +
Images: Green fluorescence comes from TetR (labeled Lysines) and red fluorescence comes from DNA (cy-5 labeled). We can clearly see that the protein amount seems to be the same, whereas the DNA fluorescence is decreased.
 +
[[File:EPFL_27-06-tetR_negctrl_A488.jpg|300px|TetR fluorescence during random DNA experiment]]
 +
[[File:EPFL_27-06-tetR_negctrl_cy5.jpg|300px|DNA fluorescence during random DNA experiment]]
 +
 +
[[File:EPFL_27-06-tetR_tetO1_A488.jpg|300px|TetR fluorescence during target DNA experiment]]
 +
[[File:EPFL_27-06-tetR_tetO1_cy5.jpg|300px|DNA fluorescence during target DNA experiment]]
 +
 +
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.
 +
 +
== Tuesday, June 28 2011 ==
 +
 +
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.
 +
 +
Henrike and Irina designed primers for both plasmids, with the goal of amplifying the following four parts:
 +
 +
 +
1) a pTet-RFP segment (820 bp)
 +
*Primer 1: J61002-Ptet-RFP-r
 +
*Primer 2: J61002-RFP-f
 +
*DNA template: Plate 1, 18 C
 +
 +
 +
2) a J61002 plasmid backbone with a terminator (2334 bp)
 +
*Primer 1: J61002-f
 +
*Primer 2: J61002-term-r
 +
*DNA template: Plate 1, 18 C
 +
 +
 +
3) a constitutive promoter for TetR (725 bp)
 +
*Primer 1: Pconst-tetR-f
 +
*Primer 2: TetR-r
 +
*DNA template: TetR Repressilator plasmid
 +
 +
 +
4) P23019 plasmid (2242 bp)
 +
*Primer 1: P23019-r
 +
*Primer 2: P23019-f
 +
*DNA template: Plate 4, 14E
 +
 +
 +
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.
 +
 +
[[File:EPFL-28-06-pcr.jpg‎|300px|TetR segment and RFP-pTet segment amplified. Both backbones unamplified]]
 +
 +
== Wednesday, 29 June 2011 ==
 +
 +
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:
 +
 +
1) P23019-r: 45.8 + 3 = 48.8
 +
 +
2) P23019-f: 60.8 + 3 = 63.8
 +
 +
3) J61002-bb-f: 52.2 + 3 = 55.2
 +
 +
4) J61002-term-r: 49.3 + 3 = 52.3
 +
 +
 +
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.
 +
 +
[[File:EPFL-29-06_J6_backb.jpg|300px|J61002 backbone amplification, but no P23019]]
 +
 +
== Thursday, 30 June 2011 ==
 +
 +
Alina and Lilia did MITOMI on tetR-GFP expressed from plasmid, 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).
 +
 +
[[File:MITOMI_30juin_tetR_consensus-negctrl_check.png|600px|GFP-tetR and cy5 fluorescence]]
 +
 +
Images: Green fluorescence comes from GFP-TetR and red fluorescence comes from DNA (cy-5 labeled).
 +
 +
This experiment confirms that wtTetR is expressed and correctly folded by SP6 expression system. The TF recognizes tetO1 consensus sequence while the tested random sequence (-)control has a significantly lower binding affinity to TetR.   
 +
 +
 +
== Gibson Assembly==
 +
 +
 +
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:
 +
 +
 +
RFP (820 bp) : 28.8 ng/microL
 +
 +
 +
J6 (2334 bp) : 26 ng/microL
 +
 +
 +
'''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.
 +
 +
We then did a transformation with these Gibson-ed plasmids and plated the cells on ampicillin plates and put them in the incubator overnight.
 +
 +
==  Gradient PCR  ==
 +
 +
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.
 +
 +
[[File:EPFL-29-06-p23019fail.jpg|300px|GFP-tetR and cy5 fluorescence]]
{{:Team:EPF-Lausanne/Templates/Footer}}
{{:Team:EPF-Lausanne/Templates/Footer}}

Latest revision as of 16:51, 9 July 2011

Retrieved from "http://2011.igem.org/Team:EPF-Lausanne/Notebook/June2011"