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

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

</biblio>

Contribution:iGEM22_WHU-China

The origin and test of Pxyl The detected protein was identified as putative xylose isomerase (XylA, SAV_7182) after the purification of cell extracts and culture supernatants. XylA expression was also detected in the presence of 1.0% D-xylose. These results suggest that the expression of the xylA gene was induced by the addition of D-xylose.

Figure 1.1 SDS-PAGE analyses protein expression in a cell extracts and b culture supernatants: 2.0% 1. Glycerol, 2. d-glucose, 3. d-xylose, 4. d-galactose, 5. d-fructose, and 6. no sugar. M: molecular weight marker. Arrows indicate Xylose isomerase (XylA) protein.

As mentioned above, XylA was induced in the presence of D-xylose. The xylA gene is located in xylose (xyl) operon, which consists of xylA (SAV_7182; xylose isomerase), xylB (SAV_7181; xylulose kinase), and xylR (SAV_7180; xylose operon regulator) genes. The xyl operon is related to D-xylose metabolism, which involves the transport of D-xylose, the isomerization of D-xylose to D-xylulose (mediated by XylA), and the phosphorylation of D-xylulose to D-xylulose-5-phosphate (mediated by XylB). XylR acts as a repressor for xyl operon, as reported in a deletion study of the xylR gene. Genes driven by xyl promoters are induced in the presence of D-xylose. xylA promoter and xyl operon were selected in order to develop a xylose-dependent protein expression system.

Figure 2

In our project, 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. 3). So to reduce the leakage of downstream protein expression, we planed to perform directed evolution.

Figure. 3 Fluorescent Microscopy of B. subtilis with pBE-XylR-Pxyl-opPet8p::sfGFP without xylose