Composite

Part:BBa_M36252:Design

Designed by: Evan Masutani, Erica Lieberman   Group: Stanford BIOE44 - S11   (2011-12-09)

Alkane Sensor Part


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 323
    Illegal EcoRI site found at 536
    Illegal PstI site found at 1052
    Illegal PstI site found at 1676
    Illegal PstI site found at 2084
    Illegal PstI site found at 2360
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 323
    Illegal EcoRI site found at 536
    Illegal NheI site found at 7
    Illegal NheI site found at 30
    Illegal PstI site found at 1052
    Illegal PstI site found at 1676
    Illegal PstI site found at 2084
    Illegal PstI site found at 2360
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 323
    Illegal EcoRI site found at 536
    Illegal BglII site found at 2037
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 323
    Illegal EcoRI site found at 536
    Illegal PstI site found at 1052
    Illegal PstI site found at 1676
    Illegal PstI site found at 2084
    Illegal PstI site found at 2360
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 323
    Illegal EcoRI site found at 536
    Illegal PstI site found at 1052
    Illegal PstI site found at 1676
    Illegal PstI site found at 2084
    Illegal PstI site found at 2360
    Illegal NgoMIV site found at 790
    Illegal NgoMIV site found at 1012
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Design of the alkane sensor is based on the work of Kok and van Beilen who respectively sequenced the alkB gene and characterized the regulation of alkB expression. alkB is the first gene in the alkBAC operon (responsible for alkane degradation) present in P. putida. The promoter of alkB (PalkB) is positively regulated by the transcription factor AlkS. AlkS binds to alkanes which diffuse into the cell. Makart demonstrated that the PalkB/AlkS system can be implemented in E. coli. Our group quantified the sensitivity of the PalkB/AlkS system to varying concentrations of various hydrocarbons.

AlkS is codon optimized to not overtax its E. coli chassis.


Similarities between alk from Pseudomonas putida and Alkanivorax borkumensis

(contribution added by Diana Bonciu, Team CNAS-Romania, 2023)

The first noticeable similarity is that the way the genes are organized in P. putida GPo1 is very similar to the organization in A. borkumensis AP1. The segment containing alkS in P. putida GPo1 is actually a homologue of the segment from the same gene in A. borkumensis AP1. The protein sequence identity between the two organisms’ proteins was between 65 and 86%. The alkS in P. putida GPo1 is evidently not as closely related to the one in A. borkumensis AP1 (only a 30% match). This suggests that, while all the other genes have similar sequences, the alkS gene originates from a different organism either in A. borkumensis AP1 or in P. putida GPo1. Another possibility is that the gene simply has evolved more rapidly, a phenomenon that has been previously observed in plasmids with xyl-genes. In the picture below the similarities between the inverted repeats in three of the genes in A. borkumensis AP1 and P. putida GPo1 can be observed.

Source

Anderson, John. "Part:BBa J23106 - Partsregistry.org." Main Page - Partsregistry.org. Partsregistry, 14 Aug. 2006. Web. 28 Oct. 2011. <https://parts.igem.org/Part:BBa_J23106>. 
 Mahajan, V.S., V.D. Marinescu, B. Chow, A.D. Wissner-Gross, and P. Carr. "Part:BBa B0034 - Partsregistry.org." Main Page - Partsregistry.org. Partsregistry, 31 Jan. 2003. Web. 28 Oct. 2011. <https://parts.igem.org/Part:BBa_B0034>.
 Lieberman E, E. Masutani.  “Part:BBa M36251 – Partsregistry.org”  Main Page - Partsregistry.org. Partsregistry, 9 Dec. 2011. Web. 10 Dec. 2011. <https://parts.igem.org/Part:BBa_M36251>.
 Rettberg, R. "Part:BBa B0010 - Partsregistry.org." Main Page - Partsregistry.org. Partsregistry, 19 Nov. 2003. Web. 28 Oct. 2011. <https://parts.igem.org/Part:BBa_B0010>.
 Lieberman E, E. Masutani.  “Part:BBa M36250 – Partsregistry.org”  Main Page - Partsregistry.org. Partsregistry, 9 Dec. 2011. Web. 10 Dec. 2011. <https://parts.igem.org/Part:BBa_M36250>.

References

Kok, M., R. Oldenhuis, M P Van Der Linden, P. Raatjes, J. Kingma, P H Van Lelyveld, and B. Witholt. "The Pseudomonas Oleovorans Alkane Hydroxylase Gene Sequence and Expression." Journal of Biological Chemistry 264.10 (1989): 5435-441.

Makart, Stefan, Matthias Heinemann, and Sven Panke. "Characterization of the AlkS/PalkB-expression System as an Efficient Tool for the Production of Recombinant Proteins InEscherichia Coli Fed-batch Fermentations." Biotechnology and Bioengineering 96.2 (2007): 326-36.

Puigbo, P. "OPTIMIZER." Evolutionary Genomics Group. Universitat Rovira I Virgili (URV), Apr. 2007. Web. 10 Dec. 2011. <http://genomes.urv.es/OPTIMIZER/>.

Van Beilen, J.B., S. Panke, S. Lucchini, A.G. Franchini, M. Rothlisberger, and B. Witholt. "Analysis of Pseudomonas Putida Alkane Degradation Gene Clusters and flanking Insertion Sequences : Evolution and Regulation of the Alk Genes." Microbiology 147 (2001): 1621-630.

Van Beilen, J. B., Marin, M. M., Smits, T. H. M., Rothlisberger, M., Franchini, A. G., Witholt, B., & Rojo, F. (2004). Characterization of two alkane hydroxylase genes from the marine hydrocarbonoclastic bacterium Alcanivorax borkumensis. Environmental Microbiology, 6(3), 264–273. doi:10.1111/j.1462-2920.2004.00567.x