Introduction

2022 SZU-China has applied a lot of genetic devices in RiceAide to treat rice sheath blight. In the process of brainstorming, we put forward the improvement for R-body gene cluster. The following is our improved composite part.

Improved R-body gene cluster

Improved part: BBa_K4286504.

We carefully investigate the properties of the R-body (Refractile inclusion bodies) and find some interesting points. The natural R-body gene cluster has four genes in the order of RebA-RebB-RebD-RebC. The wild type R-body gene cluster and respective functions of genes are shown below.

Figure 1. Wild type R-body gene cluster

Reb A has a similar structure and function to RebB, and is modified into two or more subtypes with different molecular weights before the major assembly occurs. RebA is able to bind to the elongating R-body, acting as a scaffolding protein to facilitate the assembly. As the assembly of R-body proceeds, the importance of RebA may decrease.

Reb B is the major structural subunit of R body. By isoelectric focusing electrophoresis, it was confirmed that RebB had two molecular weights, RebB of each molecular weight had six isoelectric points, and there were 12 kinds of RebB.

RebC, as a small polypeptide, may not has the post-translational function. But by binding to RebA or RebB, it may induce conformational changes in both, enabling the modifying protein to recognize and modify RebA and RebB.

It is generally believed that RebD is not transcribed or translated in E. coli. Of course it may be due to the low frequency of expression or the short expression time, so it is not recognized by tests. However, it is clear that R-body can be synthesized in E. coli without RebD.

Table 1. Functions of four genes in the R-body gene cluster

Delete RebD

First, RebD is not transcribed or translated in E. coli. Of course, it may be because the frequency of expression is very low or the expression period is short, which results in no detection. Experiments have shown that R-body can be synthesized and assembled in E. coli in the absence of RebD, indicating that RebD may not be essential for R-body production. Therefore, we propose the first improvement point, which is to delete RebD, so as to simplify the gene cluster of R-body.

Increase the transcription of RebB

Second, the main structural component of R-body is RebB, which is in the second position in the wild type gene cluster. We hope to improve the expression of RebB by replacing RebB to the first position, so as to make the synthesis and assembly of R-body in E. coli more efficient.

Figure 2. Improved R-body gene cluster

In summary, we obtained the improved R-body gene cluster, in which the genes are arranged in the order of RebB-RebA-RebC.

Verification

We compared our improved R-body gene cluster with the wild type R-body gene cluster designed by 2019 SZU-iGEM. Since the structure of R-body is complicated, it is difficult to purify them by protein labels for verification. Therefore, we characterized the effect and number of Escherichia coli treated with R-body lysis to reflect the R-body protein yield.

We treated transformant solution with 2% L-arabinose solution for 4 hours to induce R-body expression. Then we used 1 mol/L acetic acid solution to induce R-body from coiling to stretched, within which Escherichia coli were lysed. We set the following induction conditions, and measured OD 600 of E.coli solution before and after induction under each condition.

Table 2. Induction conditions of E.coli transformants

We compared Escherichia coli in LB liquid media that had induced R-body synthesis with those that had not. The following bar graph shows the decrease in OD 600 of liquid media before and after acid treatment (Fig. 3). After L-arabinose treatment, R-bodies were expressed in E.coli, which lysed a large number of E.coli after acid induction. In groups with L-arabinose treatment, OD 600 changed significantly, especially in groups RB1++ and RB2++. When E.coli were not treated with L-arabinose, R-body was absent in vivo, within which acid treatment had almost no effect on OD 600. In groups without L-arabinose treatment, only a small amount of E.coli is lysed due to the acidic environment.

Figure 3. OD 600 of E.coli In LB Liquid Media Before And After Treatments. RA1, RA2: E.coli transformants containing [RebABDC]-pRSFDuet1 plasmids. RB1, RB2: E.coli transformants containing [RebBAC]-pRSFDuet1 plasmids. ++: E.coli treated with L-arabinose and acetic acid. -+: E.coli treated with acetic acid only.

We believe that E.coli with higher R-body content lyse more thoroughly and release more cell contents, which will lead to sharper decrease in OD values. Based on the OD 600 measured before and after induction, it can be indicated that we successfully up-regulated the expression of R-body by modifying R-body gene cluster.

References

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