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Non-antibiotic selectable and counter-selectable markers:
DhlA
Background
DhlA is haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (Keuning, Janssen, & Witholt, 1985). It converts 1,2-dichloroethane (DCE) into the more toxic 2-chloroethanol and is successfully used as a counterselectable marker in plants (Naested, Fennema, Hao, Andersen, Janssen, & Mundy, 1999). This is why, we decided to assess its suitability as a counters selectable marker in bacteria.
Cloning
PCR amplification of BS-dhlA with these primers resulted in no product.
Forward primer: atga gaattc gcggccgc t tctaga gaggc tctat gataa atgc
Reverse primer: gact ctgcag cggccgc t actagt a tta t tattc tgtct cggca aagtg
Figure 14: DNA gel of PCR amplification of BS-dhlA with DhlA designed primer resulted in no bands.
Close the primers.
Characterization
Plates
To test how suitable DhlA is as counterselectable marker, BS-control and BS-dhlA strains were grown in the presence and absence of DCA. Both strains showed similar growth in presence and absence of DCA.
Figure 15.Comparison of growth of BS-contol and BS-dhlA at 0 ul and 20 ul DCA. Both strains showed similar growth in presence and absence of DCA.
Liquid cultures
Liquid cultures with different DCA concentrations of both BS-control and BS-dhlA were prepared. No distinct difference between the growths of the two strains was evident.
Figure 16. Comparison of growth of BS-contol and BS-dhlA in liquid cultures with varying DCA concentrations. Both strains showed similar growth.
Conclusion:
We extensively characterized the dehalogenase gene in plates and liquid cultures.
We determined that it is not suitable as a counterselectable marker.
Further plans:
To biobrick it.
