Difference between revisions of "Part:BBa K4182000"

Revision as of 18:53, 13 October 2022

VVDH

The gene encodes blue-light responsive protein VVD. It is a truncated VVD without N terminus 35aa.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal PstI site found at 223
12
INCOMPATIBLE WITH RFC[12]
Illegal PstI site found at 223
21
COMPATIBLE WITH RFC[21]
23
INCOMPATIBLE WITH RFC[23]
Illegal PstI site found at 223
25
INCOMPATIBLE WITH RFC[25]
Illegal PstI site found at 223
1000
COMPATIBLE WITH RFC[1000]

Profile

Name:VVD

Base Pairs:453bp

Origin:Neurospora crassa

Performed E. coli codon optimization

Usage&Biology

FIG. 1 Schematic diagram of blue light induction regulatory system

FIG. 2 Schematic diagram of blue light-induced regulation system

Research

Compared to chemical induction, the light induction is more fine-tuned and spatial controllable. In order to achieve blue-light induction, we replace the arabinose binding and dimerization domain with blue-light responsive VVD domain, generating VVD-AraC fusion protein, which will dimerization under light and promote the downstream PBAD promoter.

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 promoters for VVD-araC protein expression, and determined the best promoters by the expression yield of the green fluorescent protein. Also see more results of blue-light and VVD regulated sfGFP expression in parts BBa_K4182001 and BBa_K4182002.

FIG.3 PCR electrophoretic diagram of VVDAraC chimera gene

FIG.4 PCR electrophoretic diagram of PAVVDH-J23101 colony

FIG.5 PCR electrophoretic diagram of PAVVDH-porin colony

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.

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 ===Research===
-To realize the release of light-controlled products to achieve the goal of intelligent and sustained release, after an extensive literature survey, we found that the arabinose operon was modified to manipulate downstream gene expression, not through arabinoside chemical induction, but blue light induction[1].
+Compared to chemical induction, the light induction is more fine-tuned and spatial controllable. In order to achieve blue-light induction, we replace the arabinose binding and dimerization domain with blue-light responsive VVD domain, generating VVD-AraC fusion protein, which will dimerization under light and promote the downstream PBAD promoter.
-We chose blue light induction primarily because chemically induced gene expression systems are valuable tools for controlling biological processes for applications in basic science and biotechnology. While allowing tunability and some degree of spatial control, these systems have some limitations -they are unable to achieve complex spatiotemporal regulation [2], and often lack reversibility or require washing steps to achieve it [3]. These limitations can be overcome by using light, rather than small molecules, as external triggers. Under the light, for example, pulsating inputs that alternate between dark (off) and maximum intensity (fully on) can be produced [4] and have been shown to lead to effects not achievable with graded intensity light, such as reduced cell-to-cell variability in gene expression [5]. Indeed, the amount of cell-to-cell variation can be adjusted by adjusting the duty cycle, defined as the fraction of time that light is fully on, providing a new mode of control for studying stochasticity in gene expression[6]. This type of pulsatile input has also recently been shown to enhance the biosynthesis of products in engineered cells, enabling a new type of bioreactor operation[7]. The enzyme expression was adjusted to increase the fermentation yield [8]. This is a great help for the design of our photo-controlled herbicide production.
 ===Build===