Difference between revisions of "Team:SKLMT-China/Improve"

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<h1>Improve</h1>
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<p>For teams seeking to improve upon a previous part or project, you should document all of your work on this page. Please remember to include all part measurement and characterization data on the part page on the Registry. Please include a link to your improved part on this page.</p>
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<h3>Gold Medal Criterion #2</h3>
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<p><b>Standard Tracks:</b> Create a new part that has a functional improvement upon an existing BioBrick part. The sequences of the new and existing parts must be different. You must perform experiments with both parts to demonstrate this improvement.  Document the experimental characterization on the Part's Main Page on the Registry for both the existing and new parts. Both the new and existing Main Page of each Part’s Registry entry must reference each other. Submit a sample of the new part to the Registry.
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The existing part must NOT be from your 2018 part number range and must be different from the part documented in bronze #4.
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<b>Special Tracks:</b> Improve the function of an existing iGEM project (that your current team did not originally create) and display your achievement on your wiki.</p>
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<p>According to the problem we mentioned in the demonstration, the expression of the NicA2 protein may be toxic to<latin>E. coli</latin>so that its gene cannot be stably present in<latin> E. coli.</latin> Similarly, genes that are toxic to<latin>E. coli</latin>are difficult to achieve genetic manipulation in it, especially using replication-dependent recombination methods such as Red/ET homologous recombination. However, many proteins that are toxic to<latin>E. coli</latin>are normally present in <latin>Pseudomonas</latin>, and during the establishment of the Pf-5 promoter library of <latin>Pseudomonas fluorescens</latin>, we found that most of the promoters of P.pf-5 cannot work in<latin> E. coli.</latin><p>
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<p>Therefore, in addition to the transformation of <latin>Pseudomonas fluorescens<latin> pf-5 into a new chassis for heterologous expression, we are also interested in directly implementing genetic manipulation in <latin>Pseudomonas</latin>. The genetic manipulation of <latin>Pseudomonas</latin> was carried out earlier. The early genetic manipulation method was to use the homologous recombination of the endogenous RecA recombinase, but the efficiency was very low and it was prone to off target. In 2017, Zheng Wenzhao et al[1]. studied the construction of the <latin>Pseudomonas</latin> Red/ET recombination system and achieved preliminary results in <latin>Pseudomonas putida</latin>, <latin>Pseudomonas aeruginosa</latin>, <latin>Pseudomonas syringae</latin> and <latin>Pseudomonas fluorescens. </latin></p>
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<p>Regardless of the type of cloning, screening markers are indispensable, and our part Baa_ have improved one of the most widely used parts Baa_J04450 in iGEM. lacI is present on the genome of many host cells, which suggests that he normal expression of RFP requires the induction of IPTG and need 18 hours of waiting. We replaced the lac promoter in front of the RFP gene with the strong pf-5 promoter PampC. And hope that RFP, as a visible marker for the naked eye, can be expressed more pronouncedly and faster in in <latin>Pseudomonas fluorescens </latin>pf-5.</p>
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    <p>Reference:</p>
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<p>[1] Zheng Wenzhao. Construction and application of Red/ET recombination system in <latin>Pseudomonas</latin>[D].Shandong University, 2017</p>               
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Revision as of 15:53, 17 October 2018

  1.   Home
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Improved Parts

According to the problem we mentioned in the demonstration, the expression of the NicA2 protein may be toxic toE. coliso that its gene cannot be stably present in E. coli. Similarly, genes that are toxic toE. coliare difficult to achieve genetic manipulation in it, especially using replication-dependent recombination methods such as Red/ET homologous recombination. However, many proteins that are toxic toE. coliare normally present in Pseudomonas, and during the establishment of the Pf-5 promoter library of Pseudomonas fluorescens, we found that most of the promoters of P.pf-5 cannot work in E. coli.

Therefore, in addition to the transformation of Pseudomonas fluorescens pf-5 into a new chassis for heterologous expression, we are also interested in directly implementing genetic manipulation in Pseudomonas. The genetic manipulation of Pseudomonas was carried out earlier. The early genetic manipulation method was to use the homologous recombination of the endogenous RecA recombinase, but the efficiency was very low and it was prone to off target. In 2017, Zheng Wenzhao et al[1]. studied the construction of the Pseudomonas Red/ET recombination system and achieved preliminary results in Pseudomonas putida, Pseudomonas aeruginosa, Pseudomonas syringae and Pseudomonas fluorescens.

Regardless of the type of cloning, screening markers are indispensable, and our part Baa_ have improved one of the most widely used parts Baa_J04450 in iGEM. lacI is present on the genome of many host cells, which suggests that he normal expression of RFP requires the induction of IPTG and need 18 hours of waiting. We replaced the lac promoter in front of the RFP gene with the strong pf-5 promoter PampC. And hope that RFP, as a visible marker for the naked eye, can be expressed more pronouncedly and faster in in Pseudomonas fluorescens pf-5.

Reference:

[1] Zheng Wenzhao. Construction and application of Red/ET recombination system in Pseudomonas[D].Shandong University, 2017