Part:BBa_K4886005
Ptkt
This part is a transketolase promoter with a strong ribosomal binding site (RBS) sequence derived from Clostridium tyrobutyricum (C. tyrobutyricum).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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.
(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.
| None |