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:

  1μl pfu buffer (Fermentas)
  1μl MgSO4 (Fermentas) 
  0,5μl primers
  0,5μl dNTPs (10 mM)
  0,25μl pfu turbo polymerase
  0,5μl template DNA from Listeria
  optionally: 0,75μl DMSO

  solution was topped up with H₂O to 10μl.

• PCR programs:

inv

4min 95°C 
 (30s 95°C, 1min 45-55°C, 4min 72°C)x3 
 (30s 95°C, 1min 55-60°C, 4min 72°C)x28 
 10min 72°C 
 ~ 7°C

• Electrophoretic separation on 1% agarose gel

Results:

Making Llo fusion with secretion signal peptide

Kuba

Tasks:

• Amplification of llo(hly) for fusion with the secretion signal peptide

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.

• PCR programs:

hly

4min 95°C 
 (30s 95°C, 40s 42-46°C, 1min30s 72°C)x3 
 (30s 95°C, 40s 46-52°C, 1min30s 72°C)x28 
 10min 72°C 
 ~ 7°C

• Electrophoretic separation on 1% agarose gel

Results:

Cloning of p53 coding sequence

Marcin

Tasks:

• Transformation of chemocompetent E. coli strain DH5alpha with ligated pKS plasmid containing p53 coding sequence.
  
  Second trial

Methods:

• thaw bacteria on the ice - 10 minuts
• add 10 ul of ligation mixture to the bacteria
• incubation on the ice - 20 minutes
• heat shock - 1 minut, 42°C
• incubation on the ice - 2 minuts
• add 800 ul of SOB medium to the bacteria
• incubation in 37°C - 1 h
• Plating on selective LB medium supplemented with ampicillin, X-Gal and IPTG

Ligation was unsuccesful or there was something wrong with the plates