Part:BBa_K4182006:Design
Circuit of blue light induction regulatory system
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 3502
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 869
Illegal BamHI site found at 6543 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1230
Illegal AgeI site found at 1070
Illegal AgeI site found at 6378 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 6151
Illegal BsaI.rc site found at 3824
Illegal SapI site found at 6360
Illegal SapI.rc site found at 39
Profile
Base Pairs
6603
Design Notes
The codon of E. coli was optimized
Source
E.coli&Neurosparo ceassa
Usage&Biology
Engineer
We offer an environmentally friendly biofertilizer that attempts to solve the global ecological security and economic problems caused by the widespread use of chemical herbicides through synthetic biology. We constructed an engineered E. coli that produces aspartic acid and extracellular polysaccharide (EPS), a novel herbicide, under blue light and can be released into soil in a controlled manner at high temperatures, avoiding overuse of herbicides and possible residues, and promoting water retention and sand fixation of EPS. Our system consists of a proplasmid that converts glucose into a key precursor, GPP, and multiple functional plasmids that synthesize herbicides and EPS under blue light control. At the same time, our engineered cells would release herbicides and EPS containing lytic genes at a high temperature above 42℃. About 10% of the bacteria will escape the lysis process and recover, facilitating a new round of controlled production and release of herbicides and EPS. The intelligent synthesis and release of our biofertilizers will maximize the effects of herbicides and EPS, contributing to the environment and society.
Design
Based on the above hypothesis and the ideas provided by the literature, we designed the upstream control element of the chimeric VVD-AraC fusion structure and the downstream element to verify the effect of the modified operon. We selected sfGFP as the verification protein to efficiently test the expression of the element. The constructed circuit diagram is shown in the following figure.
FIG. 1 Verification circuit diagram of blue light-induced regulation system
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.
FIG.2 Electrophoretic diagram of porin-eSD-PCR
FIG.3 PCR electrophoretic diagram of PAVVDH-porin colony
FIG.3 Blue light induction system using plasmid vector-MCS
FIG.4 PCR electrophoretic diagram of VVDAraC chimera gene
FIG.5 PCR electrophoretic diagram of PAVVDH-J23101 colony
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