Team:Warsaw/Calendar-Main/13 July 2009

Cloning of p53 coding sequence Marcin Task 1:  Prepare PCR reaction to amplified p53 coding sequence.  Methods:  DNA template dilution:  1 &mi;l of plasmid solution (isolated in 11.07.09) was taken and diluted with 99 mi&;l of MQ water  PCR mixture composition:  proper mixture 1: 0.25 &mu;l primer 1 (50 nM; Oligo.pl) 0.25 &mu;l primer 2 (50 nM; Oligo.pl) 1.5 &mu;l dNTPs (20 &mu;M ;Fermentas) 0.5 &mu;l Pfu turbo polymerase (KNGiE) 2.5 &mu;l Pfu Turbo Buffer (Fermentas) 2.5 &mu;l MgSO4 (20 &mu;M; Fermentas) 1 &mu;l DNA template 16.5 &mu;l MQ water  proprer mixture 2: 0.25 &mu;l primer 1 (50 nM; Oligo.pl) 0.25 &mu;l primer 2 (50 nM; Oligo.pl) 1.5 &mu;l dNTPs (20 &mu;M ;Fermentas) 0.5 &mu;l Pfu turbo polymerase (KNGiE) 2.5 &mu;l Pfu Turbo Buffer (Fermentas) 2.5 &mu;l MgSO4 (20 &mu;M; Fermentas) 2 &mu;l DNA template 15.5 &mu;l MQ water  Negative control: the same as proper mixture 1, the only distinction is lack of the DNA template.</li> </ol></ul>  Program:</li> </ul> p53 (detailed destription is <a href="http://2009.igem.org/Team:Warsaw/Calendar-Main/9_July_2009">here</a> Results:  Clean-up the PCR products</li> </ul> Procedure:  DNA was purified using the A&A clean-up kit. Detailed procedure is described <a href="http://www.aabiot.com/products/dna_purification/dna_fragments/clean_up/protocol_clean_up.pdf">here</a></li> After clean-up 1 &mu;l of purified PCR product was loaded into gel and photographed.</li></ul> <img src="http://2009.igem.org/wiki/images/2/26/PCR_reaction-p53_13_07_09.png" width="45%" height="45%"> verification of PCR reaction and clean-up procedure   Comment:  Unfortunatelly reaction 1 (2 &mu;l of DNA matrix) was abortive - there is no product in the gel.  Task 2:  Restriction digest of p53 coding sequence.</li> </ul> Methods:  Control digest using PvuII</li> Reaction mixture composition: 1 &mu;l purified PCR product 0.5 &mu;l PvuII (Fermentas) 2 &mu;l Buffer Green (Fermentas) 15.5 &mu;l MQ water </li></ul> Digest for subsequent cloning using XbaI</li> <li>Reaction mixture composition: 10 &mu;l purified PCR product 1 &mu;l XbaI (Fermentas) 5 &mu;l Buffer Tango (Fermentas) 34.5 &mu;l MQ water </li></ul> <li>Both reaction were perform in the same condition:</li> </ul> Program: digest: 1. 37&deg;C - 3 hours 2. 80&deg;C - 15 minutes 3. 4&deg;C - hold <img src="http://2009.igem.org/wiki/images/6/63/P53_control_digest_13_07_09.png" heigth="50%" width="50%"> control digest of p53 with PvuII <Comment Restriction pattern confirms that sequence is correct <ul> <li>Purification of digested products via gel-out</li> </ul> Procedure: <ul> <li>Fragments of agarose gel were carefully cut out and in the next step DNA was extracted from the gel using the A&A gel-out kit. Detailed procedure is described <a href="http://aabiot.com/products/dna_purification/dna_fragments/gel_out/protocol_gel_out.pdf">here</a></li> <li>Quantification of amount of p53 DNA after restriction digest:</li> </ul> 1 &mu;l of the digest mixture was diluted to 10 &mu;l and loaded into the gel. <img src="http://2009.igem.org/wiki/images/4/47/P53_PCR_after_digest_13_07_09.png" width="50%" heigth="50%"> PCR product after digest loaded into the gel Task 3: <ul> <li>Cloning p53 coding sequence to pKS plasmid</li> </ul> Methods: <ul> <li>Ligation mixture composition: 14 &mu;l digested p53 1.5 &mu;l digested pKS 5 &mu;l ligation buffer (Invitrogen) 1 &mu;l ligase T4 </li> <li>Duration of ligation was about 12 hours</li> </ul>

Creating devices to test promoters in E. coli strains (devices BBa_K177024 and BBa_K177025)
Franek

Tasks:


 * Digestion of BBa_R0010 - lacI regulated promoter, BBa_R0080 - AraC regulated promoter and E0840 – RBS + GFP + Terminator

Methods:
 * Ligation of lacI regulated promoter and AraC regulated promoter with E0840


 * DNA containing pAraC and placI was digested with SpeI and PstI enzymes. Digestion mix contained 10&micro;l of extracted DNA, 2&micro;l of Fermentas Tango buffer, 0.5&micro;l of each enzyme and water added to obtain 20&micro;l total volume. Both mixes were incubated for 3h at 37&deg;C.


 * DNA containing E0840 was digested with XbaI and PstI enzymes. Digestion mix contained 26&micro;l of extracted DNA, 3&micro;l of Fermentas Tango buffer, 0.5&micro;l of each enzyme. Two identical mixes created this way were incubated for 3h at 37&deg;C.


 * Enzymes in all 4 mixes were inactivated through incubation at 80&deg;C for 20 min.

Results:
 * Two ligation mixes were prepared. Each contained 30&micro;l of pAraC/ placI digestion mix,  20&micro;l of E0840 mix, 5&micro;l of dNTPs and 2&micro;l of ligase. Both were left at  16&deg;C for over night incubation.


 * Positive selection will be made according to fluorescence under UV light

Making of the RBS-cI Jarek

Tasks: <ul> <li>Preparing 20 liquid cultures from colonies that grew after transformation and incubationg them in 37C degree.</li> <li>Isolation of plasmid DNA from liquid cultures.</li> <li>Digestion of isolated DNA with EcoRI and PstI endonucleases.</li>

Cloning of the mgtc promoter into the pKSII+ plasmid Kamil

Tasks: <ul> <li>Plasmid assembly</li> </ul>

Methods (the bulk technique): <ul> <li>Plasmid digest mix was prepared as follows: 2&mu;l Tango buffer (Fermentas) 3&mu;l pKSII+ plasmid 2&mu;l XbaI enzyme 2&mu;l SmaI enzyme the solution was topped up with H2O to the final volume of 20 ul.</li> <li>mgtc promoter digest mix was prepared as follows 2&mu;l Tango buffer (Fermentas) 10&mu;l purified gene 2&mu;l XbaI enzyme the solution was topped up with H2O to the final volume of 20 ul.</li> <li>The digest was kept for 3h at 37&deg;C, and then the enzyme was inactivated for 15min. at 80&deg;C.</li> <li> The ligation mix was prepared as follows: both inactivated digests were mixed together and topped with 5&mu;l of 30% PEG 5&mu;l 10mM ATP 1,2&mu;l Tango buffer (Fermentas) 2&mu;l of T4 ligase (Fermentas) The ligation was carried out in 18&deg;C overnight (~18h) and then inactivated for 10min. at 65&deg;C.</li>

Cloning of hly gene into pKSII+ vector Kama <ul> <li>chemocompetent E. coli dH5&alpha; were incubated on ice for 15 minutes</li> <li>ligation mixture was added</li> <li>bacteria were incubated with DNA on ice for 30 minutes</li> <li>heat shock was conducted (1 minute 42&deg;C)</li> <li>bacteria were incubated on ice for 3 minutes</li> <li>After the heat shock 750&mu;l of SOB medium was added</li> <li>Mixture with bacteria was incubated for 1 hour in 37&deg;C</li> <li>100&mu;l of mixture was plated on medium containing ampicillin, X-Gal and IPTG (diluted 10* and without dilution)</li> </ul> </ul> </li> </ul> </li> </ul> </li> </ul> </li> Construction of K177012 operon1_part2

Ania

Tasks:


 * Set up a liquid culture from traqnsformants containing BBa_C0012 - lacI repressor on the pSB1A2 ampicillin resistant plasmid.

BBa_C0012 - lacI repressor on the pSB1A2 ampicillin resistant plasmid.
 * Alkaline lysis of bacterial cultures to obtain plasmids containing following bricks:


 * Digest of BBa_C0012 - lacI repressor on the pSB1A2 ampicillin resistant plasmid (XbaI/PstI = prospective insert)
 * Digest of BBa_B0032 - RBS.3 on the pSB1A2 ampicillin resistant plasmid (SpeI/PstI = prospective vector)

Results:


 * Fail. Digestion mix was incubated overnight at room temperature, this might be the reason for the unproper digestion.

Isolation of BioBricks from 2008 and 2009 Kit Plates
Monika

Task 1
 * Another attempt to isolate GFP coding device switched on by IPTG - BBa_I763004 from 2008 Kit Plate 1017 well G5

Methods here)
 * 2008 Kit: isolation of DNA from selected wells (two punched paper spots) with 8ul of TE (prodedure described
 * Transformation of chemocompetent cells (prepared by Franek and Ania) with 3ul of DNA solution
 * Planting on LB-agar medium supplemented with ampicillin and kanamycin

Results
 * Will be determined tomorrow

Task 2
 * prepare culture of bacteria to isolate promoter lambda (cI regulated) with RFP reporter BBa_I763007