Difference between revisions of "Part:BBa K4886001"
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We used Pthl-adhE2 from BBa_K4408008 as the template and X-pMTL-F and X-pMTL-R as primers to obtain a X-Pthl vector (5461bp) by amplification. We used the Clostridium acetobutylicum ATCC824 genome as a template to amplify the Phosphoketolase gene [FXpk(BD)] fragment (2391bp). The vector and fragment were confirmed by gel electrophoresis (Figure 1 and 2). The FXpk(BD) fragment was ligated to the X-Pthl linear vector, using Gibson assembly. The plasmid was then transformed into E.coli JM109. After colony PCR (1000 bp) for the transformed bacterial colonies, positive colonies were inoculated, and plasmids were extracted. The recombinant plasmid pMTL-Pthl-FXpk(BD) obtained was confirmed by gene sequencing. | We used Pthl-adhE2 from BBa_K4408008 as the template and X-pMTL-F and X-pMTL-R as primers to obtain a X-Pthl vector (5461bp) by amplification. We used the Clostridium acetobutylicum ATCC824 genome as a template to amplify the Phosphoketolase gene [FXpk(BD)] fragment (2391bp). The vector and fragment were confirmed by gel electrophoresis (Figure 1 and 2). The FXpk(BD) fragment was ligated to the X-Pthl linear vector, using Gibson assembly. The plasmid was then transformed into E.coli JM109. After colony PCR (1000 bp) for the transformed bacterial colonies, positive colonies were inoculated, and plasmids were extracted. The recombinant plasmid pMTL-Pthl-FXpk(BD) obtained was confirmed by gene sequencing. | ||
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<div class="unterschrift"><b>Figure 1 Gel electrophoresis of FXpk gene from Clostridium acetobutylicum ATCC824 (2391 bp) </b> | <div class="unterschrift"><b>Figure 1 Gel electrophoresis of FXpk gene from Clostridium acetobutylicum ATCC824 (2391 bp) </b> | ||
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<div class="unterschrift"><b>Figure 2 Gel electrophoresis of X-pMTL-Pthl linear vector (5461bp) </b> | <div class="unterschrift"><b>Figure 2 Gel electrophoresis of X-pMTL-Pthl linear vector (5461bp) </b> | ||
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Revision as of 08:15, 27 September 2023
Pthl-F/Xpk(BD)
It is the part which is responsible for the expression of F/Xpk from Clostridium acetobutylicum ATCC824 with Pthl promotor. It consists of Pthl sequence (BBa_K3443002), strong ribosomal binding site (RBS) sequence (BBa_K103015), F/Xpk sequence (BBa_K4119076) and terminator sequence (BBa_K3585002). F/Xpk is a gene that encodes phosphoketolase. Phosphoketolase is an enzyme with both the Fpk and Xpk activity. It catalyzes the conversion of fructose-6-phosphate (F6P) to erythrose-4-phosphate (E4P) and acetyl-phosphate (AcP), and the conversion of xylulose-5-phosphate (X5P) to glyceraldehydes-3-phosphate (G3P) and AcP.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1986
Illegal XbaI site found at 929 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1986
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1986
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1986
Illegal XbaI site found at 929 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1986
Illegal XbaI site found at 929 - 1000COMPATIBLE WITH RFC[1000]
results
(1)Plasmid construction
We used Pthl-adhE2 from BBa_K4408008 as the template and X-pMTL-F and X-pMTL-R as primers to obtain a X-Pthl vector (5461bp) by amplification. We used the Clostridium acetobutylicum ATCC824 genome as a template to amplify the Phosphoketolase gene [FXpk(BD)] fragment (2391bp). The vector and fragment were confirmed by gel electrophoresis (Figure 1 and 2). The FXpk(BD) fragment was ligated to the X-Pthl linear vector, using Gibson assembly. The plasmid was then transformed into E.coli JM109. After colony PCR (1000 bp) for the transformed bacterial colonies, positive colonies were inoculated, and plasmids were extracted. The recombinant plasmid pMTL-Pthl-FXpk(BD) obtained was confirmed by gene sequencing.