Difference between revisions of "Team:ShanghaitechChina/Hydrogen"

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(Click to see the detail sequenced information: <a href="https://static.igem.org/mediawiki/2016/7/75/G_HydA_SpyCatcher.pdf">HydA-SpyCatcher</a>, <a href="https://static.igem.org/mediawiki/2016/d/db/G_HydA_SpyTag.pdf">HydA-SpyTag</a>, <a href="https://static.igem.org/mediawiki/2016/9/91/G_HydE.pdf">HydE</a>, <a href="https://static.igem.org/mediawiki/2016/9/98/G_HydF.pdf">HydF</a>, <a href="https://static.igem.org/mediawiki/2016/b/bf/G_HydG.pdf">HydG</a>)</p></h5>
 
(Click to see the detail sequenced information: <a href="https://static.igem.org/mediawiki/2016/7/75/G_HydA_SpyCatcher.pdf">HydA-SpyCatcher</a>, <a href="https://static.igem.org/mediawiki/2016/d/db/G_HydA_SpyTag.pdf">HydA-SpyTag</a>, <a href="https://static.igem.org/mediawiki/2016/9/91/G_HydE.pdf">HydE</a>, <a href="https://static.igem.org/mediawiki/2016/9/98/G_HydF.pdf">HydF</a>, <a href="https://static.igem.org/mediawiki/2016/b/bf/G_HydG.pdf">HydG</a>)</p></h5>
 
In particular, pACE is the “acceptor” plasmid with hydA sequence, while others are the “donor” plasmids with the auxiliary protein sequences. With one-step Cre recombination and subsequent transformation into BL21 or DH5a, we would obtain strictly fused plasmid with either all gene circuits integrated in one big plasmid or non-fused single plasmids. The screening of successful assembly involves different resistance (Ampicillin / Chloramphenicol / spectinomycin) and different kinds of origin. In pACE1, it has a replication origin that can be recognized by common DH5a or BL21. In pDC,pDS,pDk, it has a special origin (R6K gamma ori) can be recognized only by a mutation strain of <em>E. coli</em>. (PirHC or PirLC, which can express pir gene product for its replication.) Only a successful fusion into the acceptor plasmid can it propagate, using the accepters ori. Therefore, we efficiently put all four hyd sequences on one single plasmid, avoiding the potential problems imposed by the two-plasmid system.<p></p>
 
In particular, pACE is the “acceptor” plasmid with hydA sequence, while others are the “donor” plasmids with the auxiliary protein sequences. With one-step Cre recombination and subsequent transformation into BL21 or DH5a, we would obtain strictly fused plasmid with either all gene circuits integrated in one big plasmid or non-fused single plasmids. The screening of successful assembly involves different resistance (Ampicillin / Chloramphenicol / spectinomycin) and different kinds of origin. In pACE1, it has a replication origin that can be recognized by common DH5a or BL21. In pDC,pDS,pDk, it has a special origin (R6K gamma ori) can be recognized only by a mutation strain of <em>E. coli</em>. (PirHC or PirLC, which can express pir gene product for its replication.) Only a successful fusion into the acceptor plasmid can it propagate, using the accepters ori. Therefore, we efficiently put all four hyd sequences on one single plasmid, avoiding the potential problems imposed by the two-plasmid system.<p></p>
The basis of our constructs, the four sequences, are not directly obtained from bacteriaBut they are all codon-optimized to ensure high-level expression.  (The original sequences of hydrogenase are found on <a href="http://www.genome.jp">www.genome.jp.</a>)<p></p>
+
The basis of our constructs, the four sequences, are not directly obtained from bacteria. But they are all codon-optimized to ensure high-level expression.  (The original sequences of hydrogenase are found on <a href="http://www.genome.jp">www.genome.jp.</a>)<p></p>
  
 
<h3 id="CResult"> Results of cloning</h3>
 
<h3 id="CResult"> Results of cloning</h3>

Revision as of 16:40, 18 October 2016

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