Part:BBa_K4182002:Design
J23101-eSD-VVD-AraC
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 634
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 28
Illegal PstI site found at 634 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 634
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 634
- 1000COMPATIBLE WITH RFC[1000]
Profile
Base Pairs
894
Design Notes
The codon of E. coli was optimized
Source
E.coli&Neurosparo ceassa
Usage&Test
Build
According to our design, the AraC and ParaBAD genes of the Arabinose induction and regulation system from Escherichia coli and the vivid gene from Streptomyces were synthesized respectively. eSD was added as the ribosome binding site. The synthetic genes were amplified by PCR, and the gene fragments were connected by golden gate according to the circuit diagram design. We selected Native Pc, J23101, and porin as operon gene promoters, and determined the best promoters by synthesizing and detecting the final thallus concentration and the expression yield of the green fluorescent protein.
Although the J23101 promoter is not the best, we still want to share our work results.
FIG.1 PCR electrophoretic diagram of VVDAraC chimera gene
Test
It can be seen from Figures above that porin has a higher VVD transcription level and sfGFP background expression than the J23101 promoter under non-blue light induction, indicating that the porin promoter can better and more precisely initiate and regulate gene expression. FIG. 17 further proves that porin has a larger dynamic response range and better sensitivity when induced by blue light than the native PC promoter and J23101 promoter. Therefore, the PAVVDH-porin promoter was selected as the follow-up research object.
FIG.4 mRNA level of VVD and sfGFP under different promoters without blue light
FIG.5 Differential expression of green fluorescent protein of PAVVDH-Pc, PAVVDH-J2301 and PAVVDH-porin
References
[1] ROMANO E, BAUMSCHLAGER A, AKMERIÇ E B, et al. Engineering AraC to make it responsive to light instead of arabinose [J]. Nat Chem Biol, 2021, 17(7): 817-27.
[2] RAMAKRISHNAN P, TABOR J J. Repurposing Synechocystis PCC6803 UirS-UirR as a UV-Violet/Green Photoreversible Transcriptional Regulatory Tool in E. coli [J]. ACS Synth Biol, 2016, 5(7): 733-40.
[3] ONG N T, TABOR J J. A Miniaturized Escherichia coli Green Light Sensor with High Dynamic Range [J]. Chembiochem, 2018, 19(12): 1255-8.
[4] OHLENDORF R, VIDAVSKI R R, ELDAR A, et al. From dusk till dawn: one-plasmid systems for light-regulated gene expression [J]. J Mol Biol, 2012, 416(4): 534-42.