Difference between revisions of "Part:BBa K4182003"

Revision as of 20:35, 13 October 2022

Native Pc-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).

Native Pc promoter is a constitutive promoter of AraC protein in the arabinose operon of E.coli.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal PstI site found at 627
12
INCOMPATIBLE WITH RFC[12]
Illegal PstI site found at 627
21
COMPATIBLE WITH RFC[21]
23
INCOMPATIBLE WITH RFC[23]
Illegal PstI site found at 627
25
INCOMPATIBLE WITH RFC[25]
Illegal PstI site found at 627
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 Pc promoter is not the best, we still want to share our results.

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.

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.2 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.

FIG.3 The relative mRNA level of GFP of PAVVDH-Pc, PAVVDH-J2301 and PAVVDH-porin by RT-qPCR

As shown in Figure 3 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 also effective to trigger the blue-light induction.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 7)

FIG.7 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.

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K4182003 short</partinfo>
-To transform arabinose operon into blue light induction system, we selected three different promoters for comparison, and Native Pc was one of them
+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).
+Native Pc promoter is a constitutive promoter of AraC protein in the arabinose operon of E.coli.
 <!-- Add more about the biology of this part here
@@ Line 34: / Line 36: @@
 ==Usage&Test==
-===Build===
+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 Pc promoter is not the best, we still want to share our results.
-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.
+[[File:XJTU-Design1.png|500px]]
-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 Native Pc promoter is not the best, we still want to share our work results.
+FIG. 1 The blue light induced circuit
-[[File:XJTU-5.png|300px]]
+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.1 Blue light induction system using plasmid vector-MCS
-===Test===
+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.
-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-p1-hard.png|600px]]
-[[File:XJTU-bl1.png|500px]]
+FIG.2 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.
-FIG.2 mRNA level of VVD and sfGFP under different promoters without blue light
 [[File:XJTU-bl2.png|500px]]
-FIG.3 Differential expression of green fluorescent protein of PAVVDH-Pc, PAVVDH-J2301 and PAVVDH-porin
+FIG.3 The relative mRNA level of GFP of PAVVDH-Pc, PAVVDH-J2301 and PAVVDH-porin by RT-qPCR
+As shown in Figure 3 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 also effective to trigger the blue-light induction.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 7)
+[[File:XJTU-VVDp.png|600px]]
+FIG.7 Engineered cells was observed to show green fluorescence after blue-light induction
 ===References===