Up to now, we had begun with designer plasmidic constructions, i.e. to find a promoter, a sequence RBS, a reporter gene,… which can “stick” with the standard of assembly 21.

In parallel,we needed to designer primer for each gene of interest in order to amplify them by PCR. These genes of interest are the following:

• the operon hfsEFGH
• the opéron hfsABC
• the gene hfsD (separately because it is transcribed in the opposite direction ofhfs ABC and thus we would like to turn over it).
  Note: the stock of Caulobacter crescentus used is the CB15N.

Today, after discussion, we agreed to amplify hfsD (not mutation necessary), hfsEF (not mutation necessary), hfsG (to be amplified with primer carrying out the mutation wished to eliminate the site from restriction => see chart of restriction in appendix) and hfsH (to be amplified with primer carrying out the mutation wished to eliminate the site from restriction => see chart of restriction in appendix) by PCR and to ask for the synthesis of hfsABC (because it is in these 3 genes which we have the most problems on the level of the presence of undesirable sites of restriction than we should have changed by mutation). HfsABC presenting number very high of bases (approximately 2 times more than what one can normally order), it will have to be seen with “GeneArt” if it is all the same possible to have them. The budget would be approximately 1000 dollars.

Considering the fact that, excepted hfsG and hfsH, all the above mentioned genes have a homologous at E.Coli, we initially will try to see whether the adhesive could be secreted while inserting a plasmide in E.Coli containing only these 2 genes. We redrew necessary primer.

Analyze of complete genome to see what sequences are between hfsG and hfsH:

• The end of the “part hsfG” must be at the beginning of the “part hsfH” to carry out the PCR and in order to correctly be able “to stick” the pieces later (after analysis via Genbank, the two sequences overlap on the level of a hsfA).
• It would be although, in the primers, the bases to be changed are as much as possible has A or T (considering there is less of hydrogen bond to break). (see continuation of this document) (we wait green light to order them)
• To analyse the use of let us codon between hfsG and hfsH: an Excel table (bellow) was carried out on this subject. The values are expressed as a percentage.
  The two values which could pose problem are out of orange
  
  
  

It is necessary to think of phenotypical tests for our first handling . (see, for example, if the bacteria adhere to the tube culture,…). to get information about the subject (articles + I will add that in my email with Mr. Brun, autor of artcle)

• It is the sequence ORI which makes it possible to control the number of plasmids per bacterium. It will thus be necessary well to hold account of it
• to take into account the fact that it is a plasmid “low” or a “medium” or a “high” replication.

• Plac is the promoter of the opéron lactose; it is insensitive with catabolic repression.
• Ptac is an hybrid between the promoter of the opéron lactose and the opéron tryptophan. It is stronger than Plac and is sensitive to the repressor lacI.

  With regard to the promoter induced by the arabinose: AraC is a regulating protein which changes conformation in the presence of arabinose and becomes activatrice. The activation of the promoter by the arabinose thus functions only in the presence of this protein.

  A promoter induced by LacI was found:
  BBa_R0011
  
  Resistance: ampicilline
  Promoter : is compatible with standard 21 but the plasmid associated with this promoter is not compatible with the standard of assembly 21 that is a problem.

  
  
  
  Idea of “plasmid of replacement” (!!! low Copy):
  
  BBa_1716101
  
  Resisting ampicilline
  
  ORI: BBa_K112125