ULB/7 July 2009

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<h2><strong>Metting of july 7, 2009</strong></h2>
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<b>Goal</b>:</br> to transfer the system of biosynthesis of the holdfast (see article “Characterization off the Caulobacter crescentus holdfast polysaccharide biosynthesis pathway reveals significant fedundancy in the initiating glycosyltransferase and polymerase steps” page 7229” href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2580695”">http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2580695”</a> ) in E.Coli. </p>
 
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<p> The E.Coli bacterium is of ? type whereas Caulobacter crescentus is of a type. The problem when we expresse a gene of Caulobacter in E.Coli is that the signal of recognition is not the same one. It is necessary to Cloner  gene of Caulobacter without promoter and to place the cloned part  with a promoter of E.coli. </p><p>
 
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The genes which interest us are probably organized in operon. It is necessary to insert a promoter by vector. </p>
 
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We distinguishes 3 clusters different:
 
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<li> genes for the synthesis</li>
 
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<li>genes for export </li>
 
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<li>genes for the attachment </li>
 
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It is necessary to express the clusters responsible for the synthesis and export in E.Coli. we needs two plasmids (for each). Both plasmids must be able to be retorted together in the same bacterium. One of genes is expressed in the other direction (see article “Identidication off constrained required for synthesis off the adhesive holdfast in Caulobacter crescentus” page 1437, 1st part of the last diagram). This gene “will be turned over” by PCR and will be inserted in one of both plasmids previously quoted. </p><p>
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It is thus necessary:
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<li>different systems of replication for each plasmid</li>
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<li>different promoters to express differently the systems (either one or the other or both at the same time)</li>
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<li>the plasmids must have a resistance to a different antibiotic.</li>
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On the site of UNCBI, if we makes “blast” between two proteinics sequences, we obtains percentage of similarity, percentage of identity and “the univalue”. The weaker “the univalue” is, the more the sequences are similar. </p><p>
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When we add  genes on the vector, it is necessary to pay attention to proportioning. If we place a gene naturally absent at E.Coli with a promoter of E.Coli, this protein will be expressed more strongly than the remainder. It is thus more interesting to insert a block of genes. </p><p>
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Practically, it will be necessary:
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<li> a promoter (already in the vector of expression of the biobricks)
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</li><li>a sequence RBS (ribosome binding site); this sequence is rich in has and in G, it is thus necessary to find a sequence consensus at E.Coli
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</li><li>starts complementary to the bit of the lower part and starts complementary to the bit of the top
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</li><li>if we do not have a vector containing a sequence RBS, it is necessary to add a RBS on the starter
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</li><li>once the amplified gene, one it insert in the vector via sites of restriction (to be also put in the starters)
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</li><li>GFP (to make sure that the genes are actually expressed in E.Coli)
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</li><li>terminating (there is of two types at E.Coli) </li>
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To use a possible maximum of biobricks.
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To analyze the vectors:
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Stages of checking for the cloning: once the vector selected and ready to incorporate the fragment of interest, we operate a ligation. This  all the vectors do not contain?reaction does not function at 100% . Then the cells are transferred. </p><p>
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<li>- either we make a PCR controls with starters outside the amplified fragments </li>
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<li>either we extract from the plasmidic ARN (“miniprep”) </li>
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<p>We observe the size of the fragment to see if it is ok. </p>
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<b>Transcriptional fusion</b>: the gene to defer indicates the transcription activity of the promoter (its activity is proportional to the rate of transcription). </br>
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<b>Traductional fusion</b>: we mix  gene of interest with gene to defer (by removing codon stop and AUG). In this case, if the gene of interest is degraded after being expressed, the gene to defer will not be visible either.
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== July 7, 2009 ==
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As mention in Characterization of the Caulobacter crescentus Holdfast Polysaccharide Biosynthesis Pathway Reveals Significant Redundancy in the Initiating Glycosyltransferase and Polymerase Steps 1, a cluster of polysaccharide biosynthesis genes (hfsEFGH) involved in the biosynthesis of the minimum repeat unit of the holdfast polysaccharide has been identified. hfsC and hfsI genes serve to polymerization.
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Genes involved in the export and anchoring of the holdfast polysaccharide have been neglected for our project.
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[[Image:Caulobacter_glue_prots.png‎]]
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All protein names in parentheses are the E. coli homologs
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=== First idea ===
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We would like to transfer all the genes needed for a good holdfast synthesis
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As ''hfsEFGH'' and ''hfsCBAD'' gene are clusters, 2 plasmids are required :
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* one containing hfsA, B, C , D
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* the other hfsE F G H
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Both plasmids would have a different antibiotic resistance and each gene would have its own ORF.
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Primers are designed.
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The assembly standart 21 plasmid has been chosen.
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We’ll pay attention that genes do not contain any restriction enzyme cutting site corresponding to the assembly standart 21.

Revision as of 08:04, 19 August 2009

iGEM Team:ULB-Brussels Wiki


July 7, 2009

As mention in Characterization of the Caulobacter crescentus Holdfast Polysaccharide Biosynthesis Pathway Reveals Significant Redundancy in the Initiating Glycosyltransferase and Polymerase Steps 1, a cluster of polysaccharide biosynthesis genes (hfsEFGH) involved in the biosynthesis of the minimum repeat unit of the holdfast polysaccharide has been identified. hfsC and hfsI genes serve to polymerization.

Genes involved in the export and anchoring of the holdfast polysaccharide have been neglected for our project.

Caulobacter glue prots.png

All protein names in parentheses are the E. coli homologs

First idea 

We would like to transfer all the genes needed for a good holdfast synthesis As hfsEFGH and hfsCBAD gene are clusters, 2 plasmids are required :

  • one containing hfsA, B, C , D
  • the other hfsE F G H

Both plasmids would have a different antibiotic resistance and each gene would have its own ORF.

Primers are designed.

The assembly standart 21 plasmid has been chosen.

We’ll pay attention that genes do not contain any restriction enzyme cutting site corresponding to the assembly standart 21.

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