Part:BBa_K4119011
cat1 promoter Pcat1
A Constitutive Promoter from Clostridium acetobutylicum ATCC 824, with native RBS.
In our project, this promoter was used to express fnr coding sequence.
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]
Contribution of NanjingBioX 2024 team
Results
(1)Plasmid construction
Using pMTL-Pcat1 as a template, and vector-F and vector-R as primers, vector fragment was obtained by amplification. Using recombinant plasmid pET29a-BS2 as template, and Bs2-F and Bs2-R as primers, Bs2 fragment was obtained by amplification. Gibson assembly was used to ligate Bs2 fragment with the pMTL-Pcat1 linearized vector. Colony PCR was used for the transformed colonies using Pcat1-Bs2-CPF and Pcat1-Bs2-CPR as primers. The positive colonies were transferred, and the plasmids were extracted. Gene sequencing was used to verify the plasmid pMTL-Pcat1-Bs2.
Primers and Sequences
| Primers | Primer Sequences (5’-3’) |
|---|---|
| vector-F | agcttcttgaataatatttttttaacatctattttg |
| vector-R | tgaaaacttgccataaaaaccaccctttcataaat |
| Bs2-F | aagggtggtttttatggcaagttttcaaagtt |
| Bs2-R | ttaaaaaaatattattcaagaagcttttcatat |
| Pcat1-Bs2-CPF | gtagactttaaggatggaac |
| Pcat1-Bs2-CPR | ggacgtcctatttttttaac |
(2) Plasmid construction
Pcat1 is a common promoter from Clostridium acetobutylicum ATCC 824 with native RBS (BBa_K4119011). Bs2 is one of the flavin mononucleotide (FMN)-based fluorescent proteins (BBa_K4119002), which is functional under anaerobic conditions, and thus can be employed as reporters in Clostridium. pMTL-Pcat1-Bs2 recombinant plasmid is used to express Bs2 using Pcat1 as the promoter.
pMTL-Pcat1-Bs2 was transfected into Clostridium tyrobutyricum, notated as Pcat1 in figures. The transfected C. tyrobutyricum were cultured for 48h. The fermentation broth was centrifuged, washed, and resuspended in PBS, and then the diluted bacterial solution was smeared on a glass slide. By using a fluorescence microscope, green fluorescence was observed (Fig. 2).
The transfected C. tyrobutyricum (notated as Pcat1 in Figure 3) were cultured till OD600 reached 1.0, and then were taken for fluorescence intensity detection. C. tyrobutyricum strain with empty plasmid pMTL82151 was as a blank control (notated as Control). The fluorescence intensity of Pcat1 under OD600=1.0 was 21523.1. Pcat1 showed stronger intensity than Control.
| None |