Part:BBa_K4182000
VVDH
The gene encodes blue-light responsive protein VVD. It is a truncated VVD without N terminus 35aa.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 223
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 223
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 223
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 223
- 1000COMPATIBLE WITH RFC[1000]
Profile
Name:VVD
Base Pairs:453bp
Origin:Neurospora crassa
Performed E. coli codon optimization
Usage&Biology
Research
To realize the controllable synthesis and release of products, after an extensive literature survey, we found that the arabinose operon can be engineered to regulated downstream gene expression by blue light induction instead of arabinose chemical induction.
Chemically induced gene expression systems are valuable tools to control biological processes for applications in basic science and biotechnology. While for the tuned and spatial control of gene expression, chemically induced systems have some limitations-they are unable to achieve complex spatiotemporal regulation, and often lack reversibility or require washing steps to achieve it. These limitations can be overcome by using light, rather than small molecules, as external triggers. This type of pulsatile input has also recently been found to enhance the biosynthesis of products in engineered cells, enabling a new type of bioreactor operation that is much easier to handle than chemical induction. The enzyme expression was adjusted to increase the fermentation yield.
Based on the above information, 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.
Design
We designed the upstream regulator- the chimeric VVD-AraC fusion protein by replacing the arabinose binding and dimerization domain of arabinose operon with a blue-light responsive VVD domain, which will dimerization under light and promote the downstream PBAD promoter. We selected sfGFP as the reporter to verify the regulation of the system. In order to test the effect of VVD-AraC expression level on the downstream gene expression, three promoters-native Pc, J23101 and porin promoter was selected in our study (BBa_K4182001, BBa_K4182002, BBa_K4182003). The blue-light inducible circuit is shown as follows (Figure 3).
FIG. 3 The blue light-induced circuit
Build
According to our design, the VVD gene from Streptomyces were chemically synthesized, and the AraC-ParaBAD promoter in arabinose operon was amplified from Escherichia coli, and eSD from E. coli was served as the ribosome binding site. The three promoters-native Pc, J23101, and porin was obtained by PCR. All the fragments were ligated into pBBRMCS1 vector in one step via Golden Gate Assembly. Figures 4 and 5 shows the PCR fragments used for the circuit construction. The recombinant plasmids were verified by colony PCR as shown in Figure 6, which are further confirmed by sequencing. As a result, three plasmids PVVDH-Pc, PVVDH-J23101, PVVDH-porin, were successfully constructed for further test including cell growth and the expression of GFP.
Also see more results of blue-light and VVD regulated sfGFP expression in parts BBa_K4182001 and BBa_K4182002.
FIG.4 PCR result of J23101-VVD fragment
FIG.5 PCR result of porin-VVD fragment
FIG.6 Colony PCR verification of plasmid PAVVDH-porin
FIG.7 mRNA level of VVD under different promoters without blue light
FIG.8 Microscopy observation of strain harboring VVD after blur-light induction
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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