Part:BBa_K851002

pBAD-pXyl AND Gate

pBAD-pXyl is a combined promoter of D-Xylose and L-arabinose sugar sensor systems which is designed to be activated only in the presence of both sugars in the medium therefore functioning as an AND logic gate.

BIOLOGY

pXyl is an inducible promoter regulated by the transcriptional regulator XylR which, in Bacillus subtilis, regulates the expression of xyl operon[1]. Gene expression under pXyl can be induced by the addition of D-Xylose to the medium [1, 2]. The nucleotide sequence was obtained from Wilhelm, M &C. P. Hollenberg, 1985[3].

In the presence of L-arabinose, expression from pBAD is turned on while the absence of L-arabinose produces very low levels of transcription from pBAD [4, 5]. More precisely, in the absence of arabinose, the repressor protein AraC (BBa_I13458[6]) binds to the AraI1 operator site of pBAD and the upstream operator site AraO2, blocking transcription[7], but in the presence of arabinose, AraC binds to it and changes its conformation such that it interacts with the AraI1 and AraI2 operator sites, permitting transcription[7]. The nucleotide sequence was similar of that in Part:BBa_K206000[8].

For iGEM UNAM Genomics México 2012 project [9], pBAD/pXyl was used in the design of an AND logic gate[10] using a recently described new type of communication system between Bacillus Subtilis cells called Nanotubes[11].

Characterization

The characterization of this construction was made on an E.coli strain in a semi quantitative attempt. In that way, we expected that only araC was repressing the promoter because the xylR binding sites corresponds to xylR of B. subtilis. Since it requires sugars for its expression, and the system is repressed by a metabolite from the pentose metabolism pathway, we used a minimal medium, M9 (Sambrook, 1989), but we changed the glucose for arginine as carbon source in order to get the lowest interference in the expression of GFP from another monosaccharides species in the medium. We use a gradient for both, xylose and arabinose on cultures at 0.1 O.D (540 nm). We could see that the expression of GFP increases as the amount of sugars added also increases. An amount of 0.01% (g/ml) of arabinose is enough for an increase of 4 times the basal expression, and the maximum production is approximately 5 times greater for the downstream genes with 0.1%(g/ml) of arabinose. Xylose gradient had a small contribution on the expression of GFP, which could be attributed to the partial similarity of the binding sites between xylR from B. subtilis and E. coli. The measurements were made with a filter fluorometer based in three different measures for each condition.

Expression of GFP in the sugars gradient measured in fluorescence units

REFERENCES

[1] D Gartner, M Geissendorfer, & W Hillen(1988). Expression of the Bacillus subtilis xyl Operon Is Repressed at the Level of Transcription and Is Induced by Xylose J Bacteriol 170:7,3102-3109.
[2] Shamanna, D. K., and K. E. Sanderson. 1979. Genetics and regulation of D-xylose utilization in Salmonella typhimurium LT2. J. Bacteriol. 139:71-79.
[3] Wilhelm, M., and C. P. Hollenberg. 1985. Nucleotide sequence of the Bacillus subtilis xylose isomerase gene: extensive homology between the Bacillus and E. coli enzyme. Nucleic Acids Res. 13:5717-5722.
[4] Lee, N. (1980) Molecular Aspects of ara Regulation. In The Operon, J. H. Miller and W. S. Reznikoff, eds. (Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory), pp. 389-410.
[5] Lee, N., Francklyn, C., and Hamilton, E. P. (1987). Arabinose-Induced Binding of AraC Protein to araI2 Activates the araBAD Operon Promoter. Proc. Natl. Acad. Sci. USA 84, 8814-8818.
[6] https://parts.igem.org/wiki/index.php/Part:BBa_I13458
[8] https://parts.igem.org/Part:BBa_K206000
[7] Schlief, R. (2000). Regulation of the L-arabinose operon of Escherichia coli. Trends in Genetics. 16(12):559-565.
[9] http://2012.igem.org/Team:UNAM_Genomics_Mexico
[10] http://2012.igem.org/Team:UNAM_Genomics_Mexico/Project/Description
[11] Dubey GP, Ben-Yehuda S. (2011) Intercellular nanotubes mediate bacterial communication. Cell.;144(4):590-600

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 239
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 74
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 56