Part:BBa_K143057

Promoter xyl and RBS spoVG for B subtilis

Inducible promoter xyl(BBa_K143014) coupled to the strong Ribosome Binding Site spoVG(BBa_K143021) from B. subtilis.

Pxyl-spoVG can be used to take an input of xylose and give a Ribosomes per second (RiPS) output generator.

Xylose does not directly induce the expression of the promoter xyl, but requires the transcriptional regulator XylR, (BBa_K143036). This means that XylR must be constitutively expressed in B.subtilis in order to use the promoter hyper-spank as an inducible promoter. XylR is naturally expressed by B. subtilis but should be upregulated to increase efficiency.

To get the highest level of translation from this Promoter-RBS combination it must be connected to a coding region preceded by a coding region prefix1. A standard prefix will increase the distance between the RBS and the start codon, reducing translational efficiency.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

<biblio>

1. 1 https://parts.igem.org/Assembly:RBS-CDS_issues

</biblio>

Contribution:iGEM22_WHU-China

In our project, we search for an inducible promoter in Bacillus subtilis to express our SAMe transporter, and we found this part. However, the information is not enough to help us to describe such part, so we add some literature information and some our experiment results here.

We used such expression to express our SAMe transporter opPet8p. When we used such part, we found that the inhibition of XylR could be not strong enough. Without xylose induction, we can also found green fluorescent signal (Fig. 1). So to reduce the leakage of downstream protein expression, we planed to perform directed evolution.

Figure. 1 Fluorescent Microscopy of B. subtilis with pBE-XylR-Pxyl-opPet8p::sfGFP without xylose

According to literature, this xylose-induced promoter can be engineered for different application. It can be used as a genome engineering method[1], and it can be used to construct a xylose-inducible Bacillus subtilis integration[2], and so on.

Reference:
[1]Jeong DE, Park SH, Pan JG, Kim EJ, Choi SK. Genome engineering using a synthetic gene circuit in Bacillus subtilis. Nucleic Acids Res. 2015 Mar 31;43(6):e42. doi: 10.1093/nar/gku1380. Epub 2014 Dec 30. PMID: 25552415; PMCID: PMC4381049.
[2]Kim L, Mogk A, Schumann W. A xylose-inducible Bacillus subtilis integration vector and its application. Gene. 1996 Nov 28;181(1-2):71-6. doi: 10.1016/s0378-1119(96)00466-0. PMID: 8973310.