Difference between revisions of "Part:BBa K4886005"

 
 
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<partinfo>BBa_K4886005 short</partinfo>
 
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This part is a transketolase promoter with a strong ribosomal binding site (RBS) sequence derived from Clostridium tyrobutyricum (C. tyrobutyricum).
  
 
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<partinfo>BBa_K4886005 SequenceAndFeatures</partinfo>
 
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==Results==
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===(1) Plasmid construction===
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Using the recombinant plasmid Pthl-Bs2 as template and Bs2-F and Bs2-R as primers, VBs2 vector (5664bp) was amplified. Using C. tyrobutyricum genome as template, Ptkt fragment (300bp) was amplified with Ptkt-F and Ptkt-R as primers. Gibson assembly method was used to link Ptkt fragment to the VBs2 linearized vector. Colony PCR (400bp) was performed on the transformed colonies. The positive colonies were transferred and plasmid was extracted. After sequencing verification, the recombinant plasmid was obtained: pMTL-Ptkt-Bs2.
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  <img class="bild" src="https://static.igem.wiki/teams/4886/wiki/005-fig1.png">
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  <div class="unterschrift"><b>Fig.1 Construction of pMTL-Ptkt-Bs2 recombinant plasmid</b>
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===(2)Fluorescence intensity===
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By using E. coli CA434 as a donor strain, pMTL-Ptkt-Bs2 plasmid was transferred to C. tyrobutyricum (notated in figures as Ptkt). The transfected C. tyrobutyricum were cultured in RCM medium till OD600 reached 0.8 and 1.2. 1g of the bacteria were taken for fluorescence intensity detection. Pthl promoter is a strong promoter in C. tyrobutyricum. C. tyrobutyricum transfected with pMTL-Pthl-Bs2 was used as positive control (notated as Pthl). C. tyrobutyricum transfected with empty vector pMTL82151  was used as blank control (notated as Pcontrol).
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Our results showed that Ptkt failed to achieve efficient expression of Bs2 gene with detectable fluorescence intensity. The fluorescence intensity of Pthl under OD600=0.8 and OD600=1.2 is 4.28 and 4.16. Ptkt illustrated weak intensity (3.71 and 3.50) similar as the blank control.
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  <img class="bild" src="https://static.igem.wiki/teams/4886/wiki/005-fig2.png">
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  <div class="unterschrift"><b>Fig. 2 Fluorescence intensity of C. tyrobutyricum strains transfected with pMTL-Ptkt-Bs2 (Ptkt) , pMTL-Pthl-Bs2 (Pthl) and pMTL82151(Pcontrol)</b>
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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K4886005 parameters</partinfo>
 
<partinfo>BBa_K4886005 parameters</partinfo>
 
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Latest revision as of 15:02, 10 October 2023


Ptkt

This part is a transketolase promoter with a strong ribosomal binding site (RBS) sequence derived from Clostridium tyrobutyricum (C. tyrobutyricum).

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Results

(1) Plasmid construction

Using the recombinant plasmid Pthl-Bs2 as template and Bs2-F and Bs2-R as primers, VBs2 vector (5664bp) was amplified. Using C. tyrobutyricum genome as template, Ptkt fragment (300bp) was amplified with Ptkt-F and Ptkt-R as primers. Gibson assembly method was used to link Ptkt fragment to the VBs2 linearized vector. Colony PCR (400bp) was performed on the transformed colonies. The positive colonies were transferred and plasmid was extracted. After sequencing verification, the recombinant plasmid was obtained: pMTL-Ptkt-Bs2.

Fig.1 Construction of pMTL-Ptkt-Bs2 recombinant plasmid

(2)Fluorescence intensity

By using E. coli CA434 as a donor strain, pMTL-Ptkt-Bs2 plasmid was transferred to C. tyrobutyricum (notated in figures as Ptkt). The transfected C. tyrobutyricum were cultured in RCM medium till OD600 reached 0.8 and 1.2. 1g of the bacteria were taken for fluorescence intensity detection. Pthl promoter is a strong promoter in C. tyrobutyricum. C. tyrobutyricum transfected with pMTL-Pthl-Bs2 was used as positive control (notated as Pthl). C. tyrobutyricum transfected with empty vector pMTL82151 was used as blank control (notated as Pcontrol). Our results showed that Ptkt failed to achieve efficient expression of Bs2 gene with detectable fluorescence intensity. The fluorescence intensity of Pthl under OD600=0.8 and OD600=1.2 is 4.28 and 4.16. Ptkt illustrated weak intensity (3.71 and 3.50) similar as the blank control.

Fig. 2 Fluorescence intensity of C. tyrobutyricum strains transfected with pMTL-Ptkt-Bs2 (Ptkt) , pMTL-Pthl-Bs2 (Pthl) and pMTL82151(Pcontrol)