Team:Warsaw/Calendar-Main/6 July 2009

Informal meeting concerning important issues of the Project Michał, Paweł

Today in the evening we met in the lab located on Pawinskiego street to, during some transformations, discuss problematic issues of the Project. Here are some problems we have to solve:
 * how to locate whole system on 1-2 plasmids (I mean - how to prevent promotor leakage due to parallel arangement of operons?
 * new standard for cloning inverted parts?
 * or should we make just inverted parts - with exchanged prefix and suffix - it will be compatible with standard but in the opposite way :)


 * Where to put the secretion system genes?
 * and should it be inducible of constitutively expressed?


 * Secretion signal is a real problem if we want to direct our proteins to mitochondria. Both secretion and mito- signals are located in the N-terminus of protein
 * with p53 another problem is that we do not know if even with mito signal it will be located in mitochondria. Romek from ZGUW is a great source of knowledge :) :) :) and he said that proteins containing zinc fingers need to have nuclar export signal (NES) to be located outside nucleus


 * as it can be see from above point - p53 isn't ideal for induction of apoptosis
 * of course we can do some 'real research' and check what conditions are needed for p53 to function properly in that manner, but is it worth our precious holiday time?
 * we don't know, or do we? if p53, just produced by bacteria can work as the inducer of apoptosis - eukaryotic proteins could be modified in multiple ways, and p53 isn't the exception - it can be modified by phosporylation, ubiquitination, sumoilation and some other, with each modification changing its function
 * interesting alternative of p53 is Bax, which is also mitochondiral protein
 * it can be easily accesible from ZGUW (Romek again! :) )
 * we are sure that bax is inducing apoptosis and that it is a wonderful killer of eukaryotic cells
 * but we don't know if bax protein will be toxic for bacterial cells - it has an ability to kill Saccharomyces cerevisiae if overexpressed so in bacteria it can also occur


 * where to put LLO? in the invasion operon or in the endosome operon?
 * in my opinion (Paweł) it should be expressed in endosome - so the second operon
 * but Michał says that LLO need to have time for expression and escape from the endosome should be quick. Endosome operon is important especially for switching off the invasion operon

What do we have to do?? - solve these problems :) some literature search is needed

What's more - we have also planned some clonings

PCR inv Kama

Tasks:  Amplification of inv 

Methods:  PCR mixture's composition: 1&mu;l pfu buffer (Fermentas) 1&mu;l MgSO4 (Fermentas) 0,5&mu;l primers 0,5&mu;l dNTPs (10 mM) 0,25&mu;l pfu turbo polymerase 0,5&mu;l template DNA from Listeria optionally: 0,75&mu;l DMSO solution was topped up with H2O to 10&mu;l.    PCR programs: inv 4min 95&deg;C (30s 95&deg;C, 1min 45-55&deg;C, 4min 72&deg;C)x3 (30s 95&deg;C, 1min 55-60&deg;C, 4min 72&deg;C)x28 10min 72&deg;C ~ 7&deg;C   Electrophoretic separation on 1% agarose gel 

Results: <img src="http://2009.igem.org/wiki/images/4/4d/2009_07_06_inv_opisany.jpg"/>  Gel (from left)</li> </ul> <ol> control -</li> 2-6 - samples (annealing temperature increases to from the left to the right)</li> 7-11 - samples with DMSO (annealing temperature increases to from the left to the right) M - GeneRuler DNA Ladder Mix #SM0333 (Fermentas)</li></ol> Notes:  many unspecific products were obtained</li> </ul>

Making Llo fusion with secretion signal peptide Kuba

Tasks:  Amplification of llo(hly) for fusion with the secretion signal peptide</li> </ul>

Methods:

 PCR mixture's composition: 2,5ul pfu buffer (Fermentas) 2,5ul MgSO4 (Fermentas) 1,5ul primers 1,5ul dNTPs (10 mM) 0,5ul pfu turbo polymerase 1 ul of DMSO, 1ul template DNA from Listeria the solution was topped up with H2O to 25ul. </li> </ul>  PCR programs:</li> hly 4min 95&deg;C (30s 95&deg;C, 40s 42-46&deg;C, 1min30s 72&deg;C)x3 (30s 95&deg;C, 40s 46-52&deg;C, 1min30s 72&deg;C)x28 10min 72&deg;C ~ 7&deg;C

</ul>  Electrophoretic separation on 1% agarose gel</li> </ul>

Results: <img src="http://2009.igem.org/wiki/images/d/d0/Gradient3.JPG"/> <ul> <li>Gel (from left)</li> </ul> <ol> <li>M - GeneRuler DNA Ladder Mix #SM0333 (Fermentas)</li>

<li>1-7 - samples (annealing temperature increases to from the left to the right)</li> </ol> an even smaller relative amount of specific product has been obtained - a low-temperature PCR (followed by gel extraction) or reamplification appear to be necessary 

Cloning of p53 coding sequence Marcin Tasks:</P> <ul> <li>Transformation of chemocompetent E. coli strain DH5alpha with ligated pKS plasmid containing p53 coding sequence. Second trial</li> </ul> Methods: <ul> <li>thaw bacteria on the ice - 10 minuts</li> <li>add 10 ul of ligation mixture to the bacteria</li> <li>incubation on the ice - 20 minutes<li> <li>heat shock - 1 minut, 42&deg;C</li> <li>incubation on the ice - 2 minuts</li> <li>add 800 ul of SOB medium to the bacteria</li> <li>incubation in 37&deg;C - 1 h</li> <li>Plating on selective LB medium supplemented with ampicillin, X-Gal and IPTG</li></ul> Comment: Ligation was unsuccesful or there was something wrong with the plates