Difference between revisions of "Part:BBa K561001"

Latest revision as of 14:57, 12 October 2022

vgb promoter, microaerobic

vgb promoter is induced under microaerobic conditions.

Sequence and Features

Contribution From AHUT_China 2021

• Group: iGEM21_AHUT_China
• Author: Binbin Wu
• Summary: We validated an existing part of the hypoxia-inducible promoter vgb (BBa_K561001) and added the results to the corresponding BioBricks. All of this may help other teams. We hope it will make a contribution to the IGEM community.

 Based on the sequence of BBa_K561001, we demonstrated the function of vgb promoter to express the target protein in E. coli low oxygen conditions, and the results were showed as follows:

 The plasmid containing vgb promoter were constructed and transformed into E. coli BL21(DE3), then the transformed E. coli were screened using Kanamycin that was added to the LB medium. Fig. 1 showed that many colonies were grown on the plate, proving that the plasmid was transformed successfully into E.coli.

Fig 1. Transformation result of the plasmid containing vgb

 A single positive clone was cultured in LB medium overnight, and then 30μl of bacterial solution were added to 3 mL of LB medium and cultured for about 8 hours in low oxygen conditions. The whole bacteria protein and supernant protein were collected followed by Western blot identification. The results were shown in Fig. 2. It revealed that under the function of the vgb promoter, our target gene could be successfully expressed in E. coli.

Fig 2. Western blot analysis for the vectors harboring vgb promoter expressed in E. coli

In conclusion, the target protein could be induced in E. coli under the control of the vgb promoter under low oxygen conditions.

Contribution from NJTech_China 2022

Promoter vgb(BBa_K561001) was a microaerobic induced promoter of Vitreoscilla hemoglobin gene.

Considering the gene compatibility difference between different host bacteria, we designed the pMTL-Pvgb-bs2 plasmid to determine whether the promoter vgb could work normally in Clostridium tyrobutyricum by detecting the fluorescent expression intensity of fluorescent protein Bs2.

The green fluorescent protein (GFP) has been one of the most widely used reporter in bioprocess monitoring of gene expression. However, they are not functional under anaerobic conditions, and thus cannot be employed as reporters in Clostridium.Therefore, we used the Bs2 fluorescent protein, which a kind of flavin mononucleotide (FMN)-based fluorescent protein, as a reporter to verify the expression performance of the vgb promoter, and detected the intensity of the expression of fluorescence under vgb promoter in both aerobic and microaerobic conditions, respectively.

The test group: the fluorescence intensity is relatively high

The control group: the fluorescence intensity is lower compared to the test group

Improvement from iGEM22_Worldshaper-Nanjing

This year our team has upgraded vgb promoter, microaerobic (BBa_K561001) to a better version. The vgb promoter, which is a microaerobic induced promoter from Vitreoscilla hemoglobin gene with an initial FNR(fumarate-nitrate-regulator) binding site in its sequence.

Our team inserted another FNR binding site, of which the sequence was found on a research paper[1] as TTGATnnnnATCAA, 8 bp upstream of the native binding site[2] of BBa_K561001, in order to enhance the regulatory effect of FNR on vgb promoter. The regulatory effect on our improved part by oxygen concentration was expected to be more sensitive than that on BBa_K561001. By expressing the flavin mononucleotide based fluorescent protein Bs2 provided by Dr. Zhengming Zhu’s research group, the fluorescence intensity observed was used as a reference to determine whether the modified promoter met the expected effects in different oxygen concentrations. Our lab experiments showed that the improved vgb promoter was effective in E. coli CA434 transformed with pMTL-FNRBS-Pvgb-bs2 plasmid. The modified promoter showed higher inhibition on the expression of downstream genes than BBa_K561001 under both aerobic and microaerobic conditions.

Detailed experiments and results are as following. For the improved part, please refer to BBa_K4407016.

Experiments

1.Plasmid construction and transformation: PMTL-FNRBS-Pvgb-bs2

Using pMTL-Pvgb-bs2 plasmid constructed by team NJTech_China as template, site-directed mutagenesis was carried out to obtain certain megaprimers by PCR technique using well designed primer, then another PCR technique was performed on pMTL-Pvgb-bs2 with megaprimers to obtain PMTL-FNRBS-Pvgb-bs2 recombined plasmid. Plasmids obtained were transformed into E. coli DH5α competent cells and cultured positive colonies for extraction of plasmid, which was then sent for sequence analysis, and correct ones were transformed into E. coli CA434 for conjugation with Clostridium tyrobutyricum.

2.Fluorescence intensity measurement

The operations of fluorescence intensity measurement were done under the guidance of NJTech_China team members, with protocols below. The E. coli CA434 bearing pMTL-Pvgb-bs2 and pMTL-FNRBS-Pvgb-bs2 were precultured, inoculated into LB culture medium with different dissolved oxygen conditions controlled, and cultured to OD600 ~0.7 and ~1.2 respectively. Two milliliters of bacterial culture solution were taken, centrifuged at 12,000 rpm for 2min, washed with 700μl 1x PBS buffer for 2 times. After resuspending the bacterial pellet using another 700μl 1xPBS buffer, 200μl of that solution were pipetted into a 96-well plate for microplate reader analysis, in which the obtained fluorescent data were analyzed.

Results

After collation and analysis of the fluorescence intensity data, the figure was draw as below. Promoter vgb is a hypoxia-induced promoter, and it’s expected that the higher the oxygen concentration is, the stronger inhibitory effect would be. Our modified strain bearing pMTL-FNRBS-vgb-bs2 plasmid showed higher inhibition than Pvgb-bs2 under aerobic condition, which indicated that our transformation was effective. Meanwhile, the modified strain FNRBS2-Pvgb-bs2 also showed inhibitory effect on the expression of downstream genes compared with Pvgb-bs2 under microaerobic condition (Figure 1).

Figure 1. The modified promoter FNRBS-Pvgb showed higher inhibition on the expression of fluorescent protein bs2 than Pvgb in recombinant E. coli CA434 under both aerobic and microaerobic conditions.

Reference

[1]Chrystala Constantinidou, et al. A Reassessment of the FNR Regulon and Transcriptomic Analysis of the Effects of Nitrate, Nitrite, NarXL, and NarQP as Escherichia coli K12 Adapts from Aerobic to Anaerobic Growth. Journal of Biological Chemistry, Volume 281, Issue 8, 2006, Pages 4802-4815.
[2]Erin L. Mettert and Patricia J. Kiley. Reassessing the Structure and Function Relationship of the O2 Sensing Transcription Factor FNR. Antioxidants & Redox Signaling, Volume 29, Number 18, 2018.

Improvement from NJTech_China 2022

Doubling promoter vgb enhances the expression level of the promoter.

Trough literature research and preliminary experiments, we found that the expression of the vgb promoter was not ideal, so we tried to enhance its ability to express proteins by doubling promoter vgb. We decided to insert a repeated Pvgb fragment into the upstream of Pvgb , hoping that the expression of Bs2 fluorescent protein could be improved. We have converted the construction and sequence analysis towards the recombinant plasmid into E. coli CA434, but time remaining for the competition did not allow us to continue to transfer the recombined plasmid into C. tyrobutyricum by conjugation, so the detection of fluorescence intensity data were carried out in E. coli to verify the effectiveness of the improvements for the vgb promoter. By controlling aerobic and micro-aerobic culture conditions, we incubated E. coli bearing recombinant plasmid pMTL- P2vgb-bs2 together with Pvgb-bs2 as control, under those two conditions respectively, and sampled the culture solutions after logarithmic stages to detect fluorescence intensity after relevant treatment.

After collating and analyzing the fluorescence intensity data (Figure 6), it can be seen obviously that the inhibition effect of the improved promoter(P2vgb) was stronger than that of the control group(Pvgb) under aerobic conditions. Under the induction of microaerobic conditions, the improved promoter(P2vgb) behaves similarly to the control group(Pvgb) under aerobic conditions. This suggests that tandem promoters improve protein expression.

Inserting exogenous 5'-UTR sequences downstream of the promoter vgb significantly enhances the expression effects of the promoter.

Trough literature research and preliminary experiments, we found that the expression of the vgb promoter was not ideal, so we tried to enhance its ability to express proteins by the insert of a fragment of exogenous 5'-untranslated region(5'-UTR). In iGEM's official part registry catalog, we retrieved a translation enhanceing 5-UTR fragment(Part:BBa_K1758100) designed by team Bielefeld-CeBiTec in 2015, and we decided to insert this fragment downstream of the vgb promoter in the pre-constructed plasmid pMTL-vgb-bs2, hoping that the expression of the Bs2 fluorescent protein would improve. This sequence contains a 5'-UTR and a strong ribosomal binding site(RBS) from bacteriophage T7, and it's reported could greatly enhance translation of a following gene. The enhancing effect relies on the regulation of mRNA binding to and release of the ribosome S30 subunit. We have converted the construction and sequence analysis of this recombinant plasmid into E. coli CA434, but time remaining for the competition did not allow us to continue to transfer the recombined plasmid into C. tyrobutyricum by conjugation, so the detection of fluorescence intensity data was carried out in E. coli to verify the effectiveness of the improvements for the vgb promoter. By controlling aerobic and micro-aerobic culture conditions, we incubated E. coli bearing recombinant plasmid Pvgb-5'-UTR-bs2 together with Pvgb-bs2 as control, under those two conditions respectively, and sampled the culture solutions after logarithmic stages to detect fluorescence intensity after relevant treatment.

After collating and analyzing the fluorescence intensity data (Figure 8), it can be seen obviously that the improved promoter(Pvgb-5'-UTR) under aerobic conditions behaves similarly to the control group(Pvgb), while under the induction of microaerobic conditions, the expression effect of the improved promoter is 1.97-folds of the control group, which indicates that the promoter engineering strategy of inserting exogenous 5'-untranslated region(5'-UTR) does improve the expression of downstream proteins.

Adjusting the distance between FNR binding site and the -35 region of promoter vgb fine tunes the inhibitory effect of oxygen on the promoter.

By consulting the literature and consulting the authors [2], we learned that the distance between the FNR binding site and the -35 region of the promoter has a large impact on promoter transcriptional regulation. To adjust the distance of FNR binding site from the -35 region, we used site-directed mutagenesis. Megaprimer mutation technique was used to separate the FNR binding site from the -35 region by, changing the distance between the FNR binding site to 3bp and 7bp from -35 region, while changing the sequence from the second half of the FNR binding site and the -35 region to more conservative sequences [2,] with higher expressive effects, and reducing the interval speacer between-35 and-10 region to 17bp. The plasmid pMTL-Pvgb-bs2 was extracted from recombined E. coli CA434 pMTL-Pvgb-bs2 constructed previously. In this experiment, the megaprimers were obtained by PCR technique using the plasmid pMTL-Pvgb-bs2 as the template, and then the mutant plasmid was obtained by PCR using those megaprimers to amplify the plasmid.

By analyzing the fluorescence intensity data, it can be found that the increase in the distance between the FNR binding site and the -35 region of the promoter could result in a certain decrease in its expression effect. Under aerobic and microaerobic conditions, the modified promoter (Pvgb-F7) was separately 0.267 and 0.422 folds of the controlled group (Pvgb). The modified promoter still has the regulatory effect brought by FNR and its based oxygen-related biosensor system, which induction ratio increased to 6.28, compared with 3.97 of Pvgb as control.

References

[1] Ning Xu, Liang Wei, and Jun Liu; Recent advances in the applications of promoter engineering for the optimization of metabolite biosynthesis. World Journal of Microbiology and Biotechnology 2019 35:33
[2] Tingting Hao, Guohui Li, Shenghu Zhou, and Yu Deng; Engineering the Reductive TCA Pathway to Dynamically Regulate the Biosynthesis of Adipic Acid in Escherichia coli. ACS Synthetic Biology 2021 10 (3), 632-639
[3] Yi Rao, et al. Construction and Characterization of a Gradient Strength Promoter Library for Fine-Tuned Gene Expression in Bacillus licheniformis. ACS Synthetic Biology 2021 10 (9), 2331-2339
[4] Chi Cheng, et al. Development of an in vivo fluorescence based gene expression reporter system for Clostridium tyrobutyricum. Journal of Biotechnology 305 (2019) 18-22
[5] Li-Qun Jin, et al. Promoter engineering strategies for the overproduction of valuable metabolites in microbes. APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 2019
[6] Gaohua Yang, et al. Rapid Generation of Universal Synthetic Promoters for Controlled Gene Expression in Both Gas-Fermenting and Saccharolytic Clostridium Species. ACS Synth. Biol. 2017, 6, 9, 1672-1678
[7] Paweł M. Mordaka and John T. Heap; Stringency of Synthetic Promoter Sequences in Clostridium Revealed and Circumvented by Tuning Promoter Library Mutation Rates. ACS Synthetic Biology 2018 7 (2), 672-681