Part:BBa_K4182002
J23101-eSD-VVD-AraC
To transform arabinose operon into blue light induction system, we selected three different promoters for comparison, and J23101 was one of them
Sequence and Features
- 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
FIG.2 PCR electrophoretic diagram of PAVVDH-J23101 colony
Test
To explore the expression effect of synthetic plasmids, we independently design and construct a weak blue light induction system, which is mainly composed of a cold light plate and Pulse Width Modulation (PWM) modulation module, powered by USB. The size of the self-cooling plate is 20cm*20cm, the blue wavelength is 470nm, and the power is 5W/㎡.
FIG.6 Plasmid map of blue light induction regulatory system
FIG. 7 Design drawing of the self-made weak blue light induction system
Through the self-made blue light induction system, we introduced the recombinant plasmid into DH5α thallus, and successfully tested the change of green fluorescent protein yield after 4 hours of induction. Moreover, three PAVVDH promoters were compared and selected effectively.
FIG. 8 Circuit diagram of PWM regulating module
FIG. 9-10 Self-made weak blue light induction system
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.11 mRNA level of VVD and sfGFP under different promoters without blue light
FIG.12 Differential expression of green fluorescent protein of PAVVDH-Pc, PAVVDH-J2301 and PAVVDH-porin
OD600 was used to characterize the cell growth , indicating that blue light irradiation had no inhibitory effect on thallus growth, and the cell growth under induced and uninduced was consistent.
FIG.13 Line chart of OD600 absorbance value between the induced group and non-induced group
FIG.14 Line plots of sfGFP absorbance values in induced and non-induced groups
As can be seen from the above figure, compared with the non-induced group, the expression of sfGFP in bacteria undergoing the blue light induction system was significantly increased, which proved the success of our engineering construction of the blue light induction system. At the same time, PAVVD-porin as the promoter of the induction system was detected to be the best expression.
FIG. 15 VVD confocal
The results showed that fluorescent proteins were expressed in large quantities after induction, and the feasibility and efficiency of blue light induction
As shown in the following figure, a more in-depth analysis of bacterial growth and yield was conducted. The production of sfGFP in the blue-induced group was significantly improved compared with that in the blank control group. However, the calculation of the sfGFP fluorescence effect per unit volume of bacteria could more directly illustrate the efficient production capacity of the blue-induced system.
FIG. 16 Line chart of sfGFP produced per unit volume of bacteria in the induction group
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.
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