Team:Warsaw/Project

In the first step of our project we designed few BioBricks adapted for Bacillus subtilis. We decided, that even if some basic parts are available in partsregistry, we would create a few more to expand the amount of BioBricks for this bacterium. Therefore, we designed:

• Two new promoters for Bacillus subtilis
• Three new rbs for Bacillus subtilis
• One terminator for Bacillus subtilis

We believed that these parts will be useful for creating constructs designed for Bacillus subtilis in our this year's project and in the future projects based on this bacteria.

We performed all designing in Clone Manager program, which we found easy and useful.

Then we designed parts especially devoted to our project. Our goal was to create two main constructs:

First construct should allow Bacillus subtilis to enter mammalian cell. To give Bacillus this ability, we decided to use listeriolisin, protein naturally existing in Listeria monocytogenes. To confirm, that Bacillus entered the mammalian cell we used GFP protein, which would give us a fluorescent signal, easy to notice by using fluorescent microscopy.

Our plan was to test all our basic parts in this construct, by creating few parallel versions, using different promoters and rbs and choose the best version.

Our second goal was to create a plasmid which could replicate and express in both, bacterial and mammalian cell. We based our plasmid on pSB1a3 plasmid backbone.

To allow plasmid to replicate in a mammalian cell we designed special BioBrick containing oriP and EBNA1 gene from Epstein barr virus. To confirm expression of the plasmid in mammalian cell we planed to use RFP protein under control of a mammalian promoter CMV. To confirm expression in E. coli cells we used construct witch GFP.

Second main construct was devoted to use directly in mammalian cell. It was based on BioBricks from partregistry witch our new part containing oriP.
super folder GFP + CMV promoter + rbs (Kozak sequences) + RFP + oriP
We designed the oriP part especially for this construct.
Second step

• For each part we designed two starters that covered each other in about 20bp on 3' ends
• Each starter had prefix or suffix (depending on the starter) on it's 5' end
• Then we prepared PCR reaction mixture
• We used Taq polymerase mastermix (nazwa???) and primers in concentration
• We didn't add any DNA template
• We calculated the annealing temperature by oligocalculator
• For first three cycles of PCR reaction temperature was calculated for primer without overhang
• Then for thirty more cycles temperature of annealing was calculated for the whole long primer with overhang – that is because from this moment there was enough product of the reaction in the reaction mix to become a template for the next cycles
• And that's it!

Then we cloned our PCR product into pJet plasmid, using special kit for cloning PCR products (linka?), and transformed with it E. coli TOP10. After isolation of the plasmid, we confirmed the sequence of received product by DNA sequencing (traditional Sanger sequencing). From this step, our parts were ready to digest and clone with procedures recommended by iGEM :)

We managed to optimize reaction conditions and received all of ours parts by PCR reaction. Unfortunately, then we faced some problems and weren't as successful as we hoped. We didn't manage to confirm all our products' sequences by DNA sequencing. Finally we received a full BioBrick part:

• Promoter
• Two RBS
• Terminator

Third step

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As soon as we received first full and confirmed BioBrick part, we started preparing our construct for Bacillus subtilis. We began from ligation of the part with GFP, because that would allow us to check if our promoter and RBS works. We used 3A assembly procedure, witch we found on iGEM website. We followed the set pattern:



Step four

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When we had a ready construct consisting of promoter + RBS + GFP we cloned it on pTG262 vector. This plasmid, which was constructed by Edinburgh iGEM team in 2007, is the only plasmid meeting iGEM standards designed especially far Bacillus subtilis. We managed to successfully clone our insert to the vector, what we confirmed by digesting with EcoRI and PstI and gel electrophoresis of the vector and insert.