Difference between revisions of "Part:BBa K3875007"

 
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<partinfo>BBa_K3875007 short</partinfo>
 
  
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more information will be provided later.
 
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===Usage and Biology===
 
===Usage and Biology===
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<html>
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Design:
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Firstly, we selected the gene
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<i>gadB-E89Q+z452-466</i> (<a href = "https://parts.igem.org/Part:BBa_K3875000" > BBa_K3875000</a>) and
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<i>gdhA (<a href = "https://parts.igem.org/Part:BBa_K3875008" > BBa_K3875008</a>)</i>, and used the promoter
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<strong>pLlacO (<a href = "https://parts.igem.org/Part:BBa_R2011" > BBa_R0011</a>)</strong> to promote them and
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terminator <strong><a href = "https://parts.igem.org/Part:BBa_B0015" > BBa_B0015</a></strong> to terminate the procedure<strong>[1]</strong>. After that, the entire sequence
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<strong>(<a href = "https://parts.igem.org/Part:BBa_K3875007" > BBa_K3875007</a>)</strong>
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was introduced into pCS27 and yielded the recombinant plasmid <strong>pCS-lac-gadB(mut)-lac-gdhA-T1</strong>. Bellows are a picture of our plasmid profile.<br>
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<img src="https://static.igem.org/mediawiki/parts/8/8d/T--BUCT--Plasmid_profile_of_pCS-lac-gadB%28mut%29-lac-gdhA-T1.png"width="640px";height="30px"/><br>
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<strong>Method&Testing:</strong>
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Firstly, we transfer the plasimid into DH5a and culture for 12 hours, then we can pick individual colonies and shake flask to cultivate under the circumstance that the temperature is 37℃. Then we performed enzyme validation.<br>
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Meanwhile, electrophoresis of the PCR products was identified, and finally PCR product was purified by gel extraction kit. Then, we eletrotransformation the plasmid into <i>E.coli Nissele <i/>1917, and culture it for 12 hours. Then we select a single colony to continue to cultivate and prepare for fermentation.<br>
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<img src="https://static.igem.org/mediawiki/parts/1/12/T--BUCT--The_result_of_eletrotransformation.jpg"width="640px";height="30px"/><br>
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In order to detect the expression of recombinant gene, the plasmid containing  <strong> pCS-lac-gadB(mut)-gdhA</strong> was transformed into <i> E. coli</i> DH5α. for fermentation.<br>
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<strong>Testing</strong>
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We tried to verify whether the plasmid was successfully constructed. It demonstrated the success of plasmid construction.<br>
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<img src="https://static.igem.org/mediawiki/parts/4/49/T--BUCT%E2%80%94run_a_gel.png"width="640px";height="30px"/><br>
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<html>
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T--BUCT—run a gel
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Below is the result of fermentation and it shows that we have successfully producing GABA from simple carbon sources.<br>
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<img src="http://https://static.igem.org/mediawiki/parts/a/a7/T--BUCT--GABA_was_produced_by_fermentation_with_glycerol_as_substrate.png"width="640px";height="30px"/><br>
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<img src="https://static.igem.org/mediawiki/parts/8/84/T--BUCT--GABA_was_produced_by_fermentation_with_plam_oil_as_substrate.png"width="640px";height="30px"/><br>
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<img src="https://static.igem.org/mediawiki/parts/4/4f/T--BUCT--GABA_was_produced_by_fermentation_with_soybean_oil_as_substrate.png"width="640px";height="30px"/><br>
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<strong>Reference</strong>
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[1] Sheng, L., Shen, D., Yang, W., Zhang, M., Zeng, Y., Xu, J., Deng, X., & Cheng, Y. (2017). GABA Pathway Rate-Limit Citrate Degradation in Postharvest Citrus Fruit Evidence from HB Pumelo (Citrus grandis) × Fairchild (Citrus reticulata) Hybrid Population. Journal of agricultural and food chemistry, 65(8), 1669–1676. https://doi.org/10.1021/acs.jafc.6b05237
  
 
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Revision as of 08:46, 18 October 2021


Usage and Biology

Design: Firstly, we selected the gene gadB-E89Q+z452-466 ( BBa_K3875000) and gdhA ( BBa_K3875008), and used the promoter pLlacO ( BBa_R0011) to promote them and terminator BBa_B0015 to terminate the procedure[1]. After that, the entire sequence ( BBa_K3875007) was introduced into pCS27 and yielded the recombinant plasmid pCS-lac-gadB(mut)-lac-gdhA-T1. Bellows are a picture of our plasmid profile.

Method&Testing: Firstly, we transfer the plasimid into DH5a and culture for 12 hours, then we can pick individual colonies and shake flask to cultivate under the circumstance that the temperature is 37℃. Then we performed enzyme validation.
Meanwhile, electrophoresis of the PCR products was identified, and finally PCR product was purified by gel extraction kit. Then, we eletrotransformation the plasmid into E.coli Nissele 1917, and culture it for 12 hours. Then we select a single colony to continue to cultivate and prepare for fermentation.

In order to detect the expression of recombinant gene, the plasmid containing pCS-lac-gadB(mut)-gdhA was transformed into E. coli DH5α. for fermentation.
Testing We tried to verify whether the plasmid was successfully constructed. It demonstrated the success of plasmid construction.

T--BUCT—run a gel Below is the result of fermentation and it shows that we have successfully producing GABA from simple carbon sources.



Reference [1] Sheng, L., Shen, D., Yang, W., Zhang, M., Zeng, Y., Xu, J., Deng, X., & Cheng, Y. (2017). GABA Pathway Rate-Limit Citrate Degradation in Postharvest Citrus Fruit Evidence from HB Pumelo (Citrus grandis) × Fairchild (Citrus reticulata) Hybrid Population. Journal of agricultural and food chemistry, 65(8), 1669–1676. https://doi.org/10.1021/acs.jafc.6b05237 Sequence and Features BBa_K3875007 SequenceAndFeatures