Difference between revisions of "Part:BBa K4182001"

Revision as of 20:03, 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.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal PstI site found at 601
Illegal PstI site found at 866
12
INCOMPATIBLE WITH RFC[12]
Illegal PstI site found at 601
Illegal PstI site found at 866
21
COMPATIBLE WITH RFC[21]
23
INCOMPATIBLE WITH RFC[23]
Illegal PstI site found at 601
Illegal PstI site found at 866
25
INCOMPATIBLE WITH RFC[25]
Illegal PstI site found at 601
Illegal PstI site found at 866
1000
COMPATIBLE WITH RFC[1000]

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 PCR result of porin-VVD

FIG.3 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. 9-10 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.11 mRNA level of VVD under different promoters without blue light

FIG.12 The relative mRNA level of GFP of PAVVDH-Pc, PAVVDH-J2301 and PAVVDH-porin by RT-qPCR

As shown in Figure 13, 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, compare to J23101 promoter. It indicated the tight and more precise regulation by porin promoter. It is further proved in Figure 14 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.13 The cell growth of strain harboring circuit with and without blue-light induction

FIG.14 The fluorescent intensity of recombinant strain 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.

Figure 15 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 16, and significantly increased fluorescent intensity can be observed with blue light induction. The normalized fluorescent intensity (sfGFP/OD600) was also shown in Figure 17, 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 18).

FIG.16 The normalized fluorescent intensity of recombinant strain under blue-light induction

FIG. 15 VVD confocal

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.

@@ Line 36: / Line 36: @@
 ==Usage&Test==
-===Engineer===
+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).
-We offer an environmentally friendly biofertilizer that attempts to solve the global ecological security and economic problems caused by the widespread use of chemical herbicides through synthetic biology. We constructed an engineered E. coli that produces aspartic acid and extracellular polysaccharide (EPS), a novel herbicide, under blue light and can be released into soil in a controlled manner at high temperatures, avoiding overuse of herbicides and possible residues, and promoting water retention and sand fixation of EPS. Our system consists of a proplasmid that converts glucose into a key precursor, GPP, and multiple functional plasmids that synthesize herbicides and EPS under blue light control. At the same time, our engineered cells would release herbicides and EPS containing lytic genes at a high temperature above 42℃. About 10% of the bacteria will escape the lysis process and recover, facilitating a new round of controlled production and release of herbicides and EPS. The intelligent synthesis and release of our biofertilizers will maximize the effects of herbicides and EPS, contributing to the environment and society.
-===Design===
-Based on the above hypothesis and the ideas provided by the literature, we designed the upstream control element of the chimeric VVD-AraC fusion structure and the downstream element to verify the effect of the modified operon. We selected sfGFP as the verification protein to efficiently test the expression of the element. The constructed circuit diagram is shown in the following figure.
 [[File:XJTU-Design1.png|500px]]
-FIG. 1 Verification circuit diagram of blue light-induced regulation system
+FIG. 1 The blue light induced circuit
-===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.
+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.
-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.
 [[File:XJTU-4.png|300px]]
-FIG.2 Electrophoretic diagram of porin-eSD-PCR
+FIG.2 PCR result of porin-VVD
 [[File:XJTU-7.png|300px]]
-FIG.3 PCR electrophoretic diagram of PAVVDH-porin colony
+FIG.3 Colony PCR verification of plasmid PAVVDH-porin
-===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/㎡.
 [[File:XJTU-p1-map.png|400px]]
-FIG.6 Plasmid map of blue light induction regulatory system
+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.
 [[File:XJTU-p1-hard.png|600px]]
-FIG. 7 Design drawing of the self-made weak blue light induction system
+FIG. 5 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.
+[[File:XJTU-p1-hard2.png|400px]] [[File:XJTU-p1-hard3.png|400px]]
-[[File:XJTU-p1-hard1.png|400px]]
-FIG. 8 Circuit diagram of PWM regulating module
-[[File:XJTU-p1-hard2.png|400px]]
-[[File:XJTU-p1-hard3.png|400px]]
 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.
+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.
 [[File:XJTU-bl1.png|500px]]
-FIG.11 mRNA level of VVD and sfGFP under different promoters without blue light
+FIG.11 mRNA level of VVD under different promoters without blue light
 [[File:XJTU-bl2.png|500px]]
-FIG.12 Differential expression of green fluorescent protein of PAVVDH-Pc, PAVVDH-J2301 and PAVVDH-porin
+FIG.12 The relative mRNA level of GFP of PAVVDH-Pc, PAVVDH-J2301 and PAVVDH-porin by RT-qPCR
-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.
+As shown in Figure 13, 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, compare to J23101 promoter. It indicated the tight and more precise regulation by porin promoter. It is further proved in Figure 14 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.
 [[File:XJTU-p1-data1.png|500px]]
-FIG.13  Line chart of OD600 absorbance value between the induced group and non-induced group
+FIG.13  The cell growth of strain harboring circuit with and without blue-light induction
 [[File:XJTU-p1-data2.png|500px]]
-FIG.14  Line plots of sfGFP absorbance values in induced and non-induced groups
+FIG.14  The fluorescent intensity of recombinant strain 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.
-[[File:XJTU-p1-17.png|400px]]
+Figure 15 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 16, and significantly increased fluorescent intensity can be observed with blue light induction. The normalized fluorescent intensity (sfGFP/OD600) was also shown in Figure 17, 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 18).
-FIG. 15 VVD confocal
+[[File:XJTU-p1-data3.png|500px]]
-The results showed that fluorescent proteins were expressed in large quantities after induction, and the feasibility and efficiency of blue light induction
+FIG.16 The normalized fluorescent intensity of recombinant strain under 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.
+[[File:XJTU-p1-17.png|400px]]
-[[File:XJTU-p1-data3.png|500px]]
+FIG. 15 VVD confocal
-FIG. 16 Line chart of sfGFP produced per unit volume of bacteria in the induction group
 ==References==