Part:BBa_M50004:Design
alkB1
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 900
Illegal EcoRI site found at 1150 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 900
Illegal EcoRI site found at 1150 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 900
Illegal EcoRI site found at 1150
Illegal BglII site found at 976 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 900
Illegal EcoRI site found at 1150 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 900
Illegal EcoRI site found at 1150 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 643
Design Notes
This plasmid is an actuator designed to produce the alkB1 protein, the first step in degrading alkanes to clean up oil spills. We designed a plasmid with a high ORF to produce maximum copies of the alkB1 protein. The plasmid has ampicillin resistance, such that only E. Coli with a copy of this plasmid will grow on ampicillin plates. The plasmid has a T5 promoter, meaning that it is inducible by the addition of IPTG, a molecular mimic of allolactose. Thus this plasmid, once transformed into E. Coli, will produce the alkB1 protein when IPTG is present. We used a strong ribosome binding site for this plasmid because we wanted to have maximal translation of the alkB1 protein. The alkB1 gene is encoded after the ribosome binding site, the sequence of which was retrieved using the Kyoto Encyclopedia of Genes and Genomes. The sequence for alkB1 was revised to codon-optimize for E. Coli in GeneDesigner and some base pairs were modified to eliminate repeats in GeneDesigner. This gene, in tandem with the alkG gene, adds a hydroxyl group to an alkane to make it more appropriate for bacterial metabolism (Yakimov et al.). The alkB1 gene is tagged with the flag fusion tag, DYKDDDDK from DNA 2.0, in order to differentiate its expression from the alkG plasmid. The sequences for the ORF, ampicillin resistance, T5 promoter, and ribosome binding site all come from DNA 2.0.
Usage and Biology
(contribution added by Diana Bonciu and Tudor Bustan, Team CNAS-Romania, 2023)
Alcanivorax borkumensis SK2 is a marine microorganism which presumably has the ability to consume hydrocarbons such as crude oil and use them as a source of energy. This species of bacteria exists in large quantities in oil-polluted waters thanks to its ability to metabolize hydrocarbons. In Alcanivorax borkumensis, there are two documented genes that have the aforementioned property, alkB1, which can oxidize short-chain alkanes (C5-C12) and alkB2, which oxidizes medium chain alkanes(C8-C16). Both alkB1 and alkB2 are membrane proteins and also have a number of applications when it comes to hydrocarbon transformation.
In Alcanivorax borkumensis AP1
For both alkB1 and alkB2, as well as for alkS (BBa_K474000) the transcription initiation sites can, and have been, determined in past research. For the alkB1 gene, a clear signal was detected, pointing out the translation start site. Compared to this signal, the one for alkB2 was much weaker, therefore the initiation site was considered to be where the band of highest intensity was. The site was positioned 50 bp higher than the translation site.
Source
Prevalent in marine bacteria, like Alcanivorax Borkumensis.
References
“Alcanivorax Borkumensis: ABO_2707.” Kyoto Encyclopedia of Genes and Genomes. N.p., n.d. Web. 31 Oct. 2016.
Hara, A., Baik, S., Syutsubo, K., Misawa, N., Smits, T. H. M., van Beilen, J. B., & Harayama, S. (2004). Cloning and functional analysis of alkB genes in Alcanivorax borkumensis SK2. Environmental Microbiology, 6(3), 191–197. doi:10.1046/j.1462-2920.2003.00550.x
Eidani, S. Z., Shahraki, M. K., Gasemisakha, F., Hashemi, M., & Bambai, B. (2011). Cloning and expression of alkane hydroxylase-1 from Alcanivorax borkumensis in Escherichia coli. Toxicology and Industrial Health, 28(6), 560–565. doi:10.1177/0748233711416953
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