Difference between revisions of "Part:BBa K4182001"
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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.The recombinant plasmids were verified by colony PCR as shown in Figure 3. 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. | 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.The recombinant plasmids were verified by colony PCR as shown in Figure 3. 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. | ||
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| − | FIG.2 PCR result of porin-VVD | + | FIG.2-3 PCR result of porin-VVD and Colony PCR verification of plasmid PAVVDH-porin |
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FIG. 5 The self-made weak blue light induction system | FIG. 5 The self-made weak blue light induction system | ||
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FIG. 6-7 Self-made weak blue light induction system | FIG. 6-7 Self-made weak blue light induction system | ||
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FIG.12 The normalized fluorescent intensity of recombinant strain under blue-light induction | FIG.12 The normalized fluorescent intensity of recombinant strain under blue-light induction | ||
| − | [[File:XJTU-VVDp.png| | + | [[File:XJTU-VVDp.png|600px]] |
FIG.13 Engineered cells was observed to show green fluorescence after blue-light induction | FIG.13 Engineered cells was observed to show green fluorescence after blue-light induction | ||
Latest revision as of 20:14, 13 October 2022
Porin-eSD-VVD-AraC
To realize the controllable synthesis and release of products, we developed the blue-light inducible system by replacing the arabinose binding and dimerization domain of arabinose operon with blue-light responsive VVD domain, generating VVD-AraC fusion protein, 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).
Porin promoter is identified from a halophile (Halomonas sp.TD01), a constitutive promoter with a high efficiency.
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Profile
Base Pairs
1076
Design Notes
Codon optimization based on E. coli
Source
E.coli&Neurosparo crassa
Usage&Test
Based on the above information, 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 1).
FIG. 1 The blue light induced circuit
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.The recombinant plasmids were verified by colony PCR as shown in Figure 3. 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.
FIG.2-3 PCR result of porin-VVD and Colony PCR verification of plasmid PAVVDH-porin
FIG.4 Map of plasmid PAVVDH-porin
To test expression of sfGFP of the three plasmids, we developed a weak blue light induction system, which is mainly consist of a blue light plate and Pulse Width Modulation (PWM) module powered by USB. The size of the light plate is 20cm*20cm, the blue wavelength is 470nm. As the intensity of the commonly used blue light is higher than what we need in our experiment, the PWM module was employed here to adjust the intensity of light to about 5W/m2.
FIG. 5 The self-made weak blue light induction system
FIG. 6-7 Self-made weak blue light induction system
The recombinant DH5a cells harboring the blue-light inducible plasmids were cultivated at 37℃ to OD600=0.6-0.8, then cells were exposed to the self-made blue light induction system for 4 hours, and the control ones without blue-light were covered by aluminium foil. The cell density (OD600) and the fluorescent intensity of sfGFP were detected every 1 h. The results are shown as follows.
FIG.8 mRNA level of VVD under different promoters without blue light
FIG.9 The relative mRNA level of GFP of PAVVDH-Pc, PAVVDH-J2301 and PAVVDH-porin by RT-qPCR
As shown in Figure 8, without blue-light induction, a higher VVD transcription level was observed when porin promoter was used to control the expression of VVD-AraC fusion protein, compared to J23101 promoter. It indicated the tight and more precise regulation by porin promoter. It is further proved in Figure 9 that porin promoter exhibited a higher fluorescence, a wider dynamic range and better sensitivity when induced by blue light than the native PC promoter and J23101 promoter. Therefore, the plasmid PAVVDH-porin was selected for our further studies.
FIG.10 The cell growth of strain harboring circuit with and without blue-light induction
FIG.11 The fluorescent intensity of recombinant strain in induced and non-induced groups
Figure 10 showed the similar cell growth of recombinant strain with PAVVDH-porin circuit under blue light or no blue light, indicating no growth inhibition of blue-light. However, the expression level of sfGFP varied in induced-group and non-induced group as shown in Figure 11, and significantly increased fluorescent intensity can be observed with blue light induction. The normalized fluorescent intensity (sfGFP/OD600) was also shown in Figure 12, which revealed a constant increase of normalized sfGFP with time, further directly illustrating the efficient induction capacity of the blue-light induction system. The induction effect of blue-light was also confirmed by confocal, and after blue-light induction, numerous cells with green fluorescence were observed in the microscopy (Figure 13).
FIG.12 The normalized fluorescent intensity of recombinant strain under blue-light induction
FIG.13 Engineered cells was observed to show green fluorescence after blue-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. [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.