Difference between revisions of "Part:BBa K561001"

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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.

Improvement

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.

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.