Part:BBa_K4286504

Improved version of Refractile inclusion body gene cluster

We carefully examine the properties of 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. Reb A can act as a scaffolding protein to facilitate the major polymerization process; Reb B is the major structural subunit of the R body; a polypeptide as small as RebC, by binding to RebA or RebB, induces a conformational change that enables the modifying protein to modify RebAB; and RebD is not transcribed or translated in E. coli. First of all, RebD is not transcribed or translated in E. coli, of course, it may be due the frequency of expression is very low or only expressed for a short time, it is not recognized by the test. Experiments have shown that R-body can be synthesized in E. coli in the apparent absence of RebD,indicating that RebD may not be essential for R-body production. Therefore, we propose the first improvement, which is to delete RebD to ensure the simplification of the gene cluster of R-body; Secondly, the main structural component of our R-body protein complex is RebB, and RebB is in the second place in the natural gene cluster. We plan to move RebB to the first place., so as to improve the expression of RebB, and make the synthesis and assembly of R-body in E. coli more efficient. By regulating pH, R-body extension can physically disrupt the entire body of the bacterium, thereby releasing shRNA molecules transcribed by E. coli. In summary, we obtained a modified version of the R-body gene cluster, in which the gene are arranged in the order of RebB-RebA-RebC.

Figure 1. R-body gene cluster before and after modification

Assembly

We constructed the recombinant vectors [RebABDC]-pRSFDuet1 and [RebBAC]-pRSFDuet1, through which we compared the R-body production between the part of 2019 SZU-China and our improved part.

Figure 2. Plasmid [RebABDC]-pRSFDuet1 (original version of R-body gene cluster) Figure 3. Plasmid [RebBAC]-pRSFDuet1 (original version of R-body gene cluster)

We transferred the recombinant vector into E.coli Ht115(DE3). Transformants were clearly visible on the culture medium after 16 hours of incubation at 37℃.

Figure 4. Colonial morphology of E.coli transformants (left: [RebABDC]-pRSFDuet1 transformants; right: [RebBAC]-pRSFDuet1 transformants).

We selected 8 single colonies on each culture medium and carried out colony PCR for plasmid amplification. The theoretical length of the amplified product was 1730bp (RebABDC) and 1340bp (RebBAC). Electrophoresis was performed in a 1% agarose gel. The results showed that all the colonies were positive transformants, which indicated that the recombinant vector was successfully transformed.

Figure 5. 1% agarose gel stained with RebABDC and RebBAC integration of pRSFDuet1 in E.coli Ht115(DE3) was checked by PCR. The capitalized word with a number represents the sample we choose. M: 2000bp Marker. RA1~8: RebABDC transformants. RB1~8: RebBAC transformants.

After enlarged production of recombinant plasmids, we conducted double restriction enzyme digestion for further verification. Restriction enzyme NdeI & HindIII was used for digestion. For [RebABDC]-pRSFDuet1, the theoretical sizes of bands are 449bp and 4094bp; for [RebBAC]-pRSFDuet1, the theoretical sizes of bands are 98bp and 4196bp. Electrophoresis was performed in a 1% agarose gel. The results showed successful double-enzyme digestion and correct plasmid extraction.

Figure 6. 1% agarose gel stained with RebABDC and RebBAC integration of pRSFDuet1 in E.coli Ht115(DE3) was checked by restriction enzyme digestion. M: 10000bp Marker. pRA1, pRA2, pRB1, pRB2: the plasmids without enzyme digestion, which showed closed circular plasmid DNA (cc DNA) and open circular plasmid DNA (oc DNA). s-pRA1, s-pRA2, s-pRB1, s-pRB2: plasmid digested by restriction enzyme HindIII, which showed linear plasmid DNA (Linear DNA) with theoretical band size 4543bp and 4294bp for RA and RB, respectively. d-pRA1, d-pRA2, d-pRB1, d-pRB2: plasmid digested by restriction enzyme NdeI and HindIII, which showed theoretical band size 449bp and 4094bp for RA, 98bp and 4196bp for RB.

Characterization

We compared our improved R-body part with the former R-body part 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 unwrapping, within which Escherichia coli can be lysed. We set the following induction conditions, and measured OD 600 of E.coli solution before and after induction under each condition.

Table 1. Induction conditions of E.coli transformants

We compared Escherichia coli in LB liquid media that had induced R-body production with those that had not. The following bar graph shows the decrease in OD 600 of liquid media before and after acid treatment (Fig. 7). After L-arabinose treatment, R-body were produced 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 died, which was due to acidic environment. It can be indicated that we successfully induced R-body production and E.coli lysis, using L-arabinose and acetic acid, respectively.

Figure 7. 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.

Sequencing

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1207
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1042
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 1024
  

Reference

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[2]Wang B, Lin YC, Vasquez-Rifo A, Jo J, Price-Whelan A, McDonald ST, Brown LM, Sieben C, Dietrich LEP. Pseudomonas aeruginosa PA14 produces R-bodies, extendable protein polymers with roles in host colonization and virulence. Nat Commun. 2021 Jul 29;12(1):4613. doi: 10.1038/s41467-021-24796-0. PMID: 34326342; PMCID: PMC8322103.

[3]Koehler L, Flemming FE, Schrallhammer M. Towards an ecological understanding of the killer trait - A reproducible protocol for testing its impact on freshwater ciliates. Eur J Protistol. 2019 Apr;68:108-120. doi: 10.1016/j.ejop.2019.02.002. Epub 2019 Feb 12. PMID: 30826731.