Difference between revisions of "Part:BBa K4192022"

Latest revision as of 14:30, 10 October 2022

FpOAR, oxalate efflux channel protein

It can discharge oxalate form thallus, so that the bacteria can adapt to the high concentration of oxalic acid environment. It is original from Fomitopsis palustris strain TYP6137, and we have tested it in E.coli and Pseudomonas fluorescens.

Usage

Basic part FpOAR, encoding the efflux channel of oxalate.In our project, we used this part to efflux oxalic acid produced by engineering bacteria to achieve extracellular enrichment of oxalic acid. After exogenously adding calcium ions, calcium oxalate protective film was formed.

Biology

Characterization

The part comes from Fomitopsis palustris originally, therefore we need to test if the gene FpOAR can work in prokaryotes as well.

For the engineered bacteria with the FpOAR, we expected that it can have stronger viability in medium with oxalate than the control group. Bacteria can spontaneously absorb oxalate in the medium, but oxalate is harmful to bacteria because it chelates calcium ions. Consequently, engineered bacteria with the FpOAR can efflux oxalate, ends up with a better survival state. To perform this assay, we designed a gene circuit to insert FpOAR into pUC18, a commonly used vector in E. coli. As for control, we use the E. coli which has transferred an empty pUC18 vector.

We built the genetic circuit using One Step Cloning Kit II (Vazyme Biotech, China) to connect RBS sequence (BBa_K4192000) upstream to BBa_K4192022. We successfully constructed the above sequences and verified them by colony PCR and sequencing.

We tested the gene by simple quantitive test of the concentration of bacteria in the liquid medium by testing the OD595 of the bacterial solution after 12 hours of culture and induction.

Fig.1 Result of FpOAR test in E. coli, the control is negative control with empty vector.

Compared with OA-control group, the OA-experimental group significantly showed better ability to survive in oxalic acid, especially in 2 mM and 4 mM oxalate. And the effect of pH when the concentrate is 2 mM can be ignored, indicating that FpOAR can efflux OA. However, the OA-experimental group did not grow well in the higher concentration of oxalic acid. And it is speculated that the higher pH caused by higher concentration of oxalic acid obscured the differences between the groups in HCl, leading to the HCl-experimental group and the HCl-control group could not survive in it. At the same time, the experimental method of accessing single colony is not rigorous enough.

We designed a qualitative test using LB-agar medium to better characterize this part in our chassis P. fluorescens 2P24. Then we use the growth of bacteria to judge the effectiveness of the part. It is difficult to measure and adjust the pH of solid media. But when we retraced the meaning of adjusting pH to set the hydrochloric acid group, we found that this experiment essentially required different concentrations of oxalate. Therefore, we used sodium oxalate, oxalic acid and sodium hydroxide to prepare mother liquor which pH=7 and oxalate concentration of 0.1 M, so as to prepare media containing different oxalate concentrations. In this way, we can rule out the influence of low pH on the growth of bacteria and the experimental results.

In order to adapt to the change of chassis bacteria, we changed the vector to pBBR1MCS2. After the bacteria had been cultured at 28 ℃ for 12 hours, the FpOAR was induced by IPTG.

After the medium were prepared, we added the same amount of induced bacterial solution to each of the medium, and they were first adjusted to the same OD595 with ddH2O. The control group is P. fluorescens 2P24 with an empty pBBR1MCS2 vector. After 2 days, the medium shows expected result.

Fig.2 A: the experiment group, B: the control group (P.fluorescens 2P24 WT), the numbers on the lower right corner is concentrate of added oxalate

This result show that in the oxalate concentration range of 0-10 mM, the growth of the experimental groups did not differ and was not affected. However, the growth state of the control group decreased apparently with the increase of oxalate concentration, except that it grew well on the medium of 0 mM oxalate. Although a small number of bacteria survived on 2 mM and 4 mM of oxalate, the higher concentration of oxalate medium could be said to show no signs of survival of the control bacteria.

We can conclude that BBa_K4192022 is able to excrete oxalate that works both in E. coli and P. fluorescens. Its strength tends to be stronger in P. fluorescens, but that conclusion may also have been influenced by the way the experiment was conducted.

Sequence and Features