Difference between revisions of "Part:BBa K4182002"
 
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<partinfo>BBa_K4182002 short</partinfo>
 
<partinfo>BBa_K4182002 short</partinfo>
  
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To transform arabinose operon into blue light induction system, we selected three different promoters for comparison, and J23101 was one of them
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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).
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
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==Usage&Test==
 
==Usage&Test==
  
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===Build===
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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). Although the J23101 promoter is not the best, we still want to share our results.
  
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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.
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[[File:XJTU-Design1.png|500px]]
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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.
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Although the J23101 promoter is not the best, we still want to share our work results.
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FIG. 1 The blue light induced circuit
  
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[[File:XJTU-3.png|300px]]
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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.
  
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FIG.1 PCR electrophoretic diagram of VVDAraC chimera gene
 
  
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[[File:XJTU-6.png|300px]]
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[[File:XJTU-3.png|300px]]  [[File:XJTU-6.png|330px]]
  
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FIG.2 PCR electrophoretic diagram of PAVVDH-J23101 colony
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FIG.2-3 PCR result of J23101-VVD fragment and colony PCR verification of plasmid PAVVDH-J23101
  
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===Test===
 
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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/㎡.
 
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[[File:XJTU-p1-map.png|400px]]
 
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FIG.6 Plasmid map of blue light induction regulatory system
 
  
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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.
  
 
[[File:XJTU-p1-hard.png|600px]]
 
[[File:XJTU-p1-hard.png|600px]]
  
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FIG. 7 Design drawing of the self-made weak blue light induction system
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FIG.4 The self-made weak blue light induction system
  
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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.
 
  
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[[File:XJTU-p1-hard1.png|400px]]
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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.
  
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FIG. 8 Circuit diagram of PWM regulating module
 
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[[File:XJTU-p1-hard2.png|400px]]
 
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[[File:XJTU-p1-hard3.png|400px]]
 
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FIG. 9-10 Self-made weak blue light induction system
 
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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.
 
  
 
[[File:XJTU-bl1.png|500px]]
 
[[File:XJTU-bl1.png|500px]]
  
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FIG.11 mRNA level of VVD and sfGFP under different promoters without blue light
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FIG.5 mRNA level of VVD under different promoters without blue light
  
 
[[File:XJTU-bl2.png|500px]]
 
[[File:XJTU-bl2.png|500px]]
  
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FIG.12 Differential expression of green fluorescent protein of PAVVDH-Pc, PAVVDH-J2301 and PAVVDH-porin
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FIG.6 The relative mRNA level of GFP of PAVVDH-Pc, PAVVDH-J2301 and PAVVDH-porin by RT-qPCR
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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.
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[[File:XJTU-p1-data1.png|500px]]
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FIG.13  Line chart of OD600 absorbance value between the induced group and non-induced group
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[[File:XJTU-p1-data2.png|500px]]
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FIG.14  Line plots of sfGFP absorbance values in induced and non-induced groups
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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.
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[[File:XJTU-p1-17.png|400px]]
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FIG. 15 VVD confocal
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The results showed that fluorescent proteins were expressed in large quantities after induction, and the feasibility and efficiency of blue light induction
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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.
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[[File:XJTU-p1-data3.png|500px]]
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As shown in Figure 5, 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 6 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, but J231010 promoter is still a good trial.
  
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FIG. 16 Line chart of sfGFP produced per unit volume of bacteria in the induction group
 
  
 
===References===
 
===References===

Latest revision as of 20:38, 13 October 2022


J23101-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).

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 634
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 28
    Illegal PstI site found at 634
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 634
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 634
  • 1000
    COMPATIBLE WITH RFC[1000]


Profile

Base Pairs

894

Design Notes

The codon of E. coli was optimized

Source

E.coli&Neurosparo ceassa

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). Although the J23101 promoter is not the best, we still want to share our results.

XJTU-Design1.png

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.


XJTU-3.png XJTU-6.png

FIG.2-3 PCR result of J23101-VVD fragment and colony PCR verification of plasmid PAVVDH-J23101


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.

XJTU-p1-hard.png

FIG.4 The 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.


XJTU-bl1.png

FIG.5 mRNA level of VVD under different promoters without blue light

XJTU-bl2.png

FIG.6 The relative mRNA level of GFP of PAVVDH-Pc, PAVVDH-J2301 and PAVVDH-porin by RT-qPCR

As shown in Figure 5, 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 6 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, but J231010 promoter is still a good trial.


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.