Difference between revisions of "Part:BBa K4182001:Design"

Revision as of 14:26, 10 October 2022

Porin-eSD-VVD-AraC

Profile

Base Pairs

1076

Design Notes

The codon of E. coli was optimized

Source

E.coli&Neurosparo crassa

Usage&Test

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

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. [5] LI X, ZHANG C, XU X, et al. A single-component light sensor system allows highly tunable and direct activation of gene expression in bacterial cells [J]. Nucleic Acids Res, 2020, 48(6): e33.