Difference between revisions of "Part:BBa K3875007"
 
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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>
 
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>
 
<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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<strong>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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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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Testing
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We tried to verify whether the plasmid was successfully constructed. It demonstrated the success of plasmid construction.</strong><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>
 
<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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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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<strong> Below is the result of fermentation and it shows that we have successfully producing GABA from simple carbon sources.<br>
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GABA was produced by fermentation with glycerol as substrate</strong>
  
 
<img src="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>
 
<img src="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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<strong> GABA was produced by fermentation with plam oil as substrate</strong>
 
<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>
 
<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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<strong> GABA was produced by fermentation with soybean oil as substrate</strong>
 
<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>
 
<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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Based on these statistics, it is obvious that we have successfully <b>produce<b> GABA. Also, considering that normal synthesis pathways, which consisted of <b>gadB(wild)<b>, of producing GABA usually produce GABA for <b>1mg/ml<b>, so it is obvious that our gadB(mut) gene plays its special function and helps overproducing GABA！<br>
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<strong>Reference</strong>
 
<strong>Reference</strong>
 
[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
 
[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

Latest revision as of 02:57, 19 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.









Below is the result of fermentation and it shows that we have successfully producing GABA from simple carbon sources.
GABA was produced by fermentation with glycerol as substrate




GABA was produced by fermentation with plam oil as substrate




GABA was produced by fermentation with soybean oil as substrate


Based on these statistics, it is obvious that we have successfully produce GABA. Also, considering that normal synthesis pathways, which consisted of gadB(wild), of producing GABA usually produce GABA for 1mg/ml, so it is obvious that our gadB(mut) gene plays its special function and helps overproducing GABA！




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