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From 2013.igem.org

Project Registry Parts

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Gibson

Gibson Assembly *should* make for a much easier, simpler, rapid assembly of different genes than conventional PCR and cloning, plus there’s much more flexibility for optimisation through gene synthesis.

• The assembly works on fragments of DNA with ~30bp of overlapping sequence, which is exposed as 5’ single-stranded overhangs by an exonuclease. A ligase joins the overlapping regions and a polymerase fills in any gaps left by the exonuclease. These enzymes can all work together in a single reaction tube with many different overlapping fragments, making the assembly a very, very simple activity. Gibson Assembly is based on the older technique of [http://nar.oxfordjournals.org/content/32/12/e98.full| PCR Assembly], with the similar reliance on the initial construction of 200+bp fragments from smaller oligos, but with a greater degree of sequence fidelity due to less polymerase activity.
• Here’s a great introduction from IDT’s magazine, [http://www.idtdna.com/pages/decoded/decoded-articles/core-concepts/decoded/2012/01/10/isothermal-assembly-quick-easy-gene-construction| DECODED], and a more in-depth webinar.
• If you’re historically-minded or want more detail, try the [http://diyhpl.us/~bryan/papers2/bio/venter/Enzymatic%20assembly%20of%20DNA%20molecules%20up%20to%20several%20hundred%20kilobases.pdf| original paper] in which Gibson Assembly was described - or one of the coolest and most famous applications of Gibson, building a [http://www.ncbi.nlm.nih.gov/books/NBK84435/| synthetic genome].

Design

Choice of genes

Mox, chloroethanol dehydrogenase

• In sketches of our project (February-March), we planned to use Mox as the alcohol dehydrogenase converting chloroethanol to chloroacetaldehyde. After a little more research we discovered that this enzyme requires the co-factor PQQ, and unfortunately for us, this co-factor cannot be synthesised by E. Coli and would have required us to add at least one more gene to our final construct (eg, PQQ synthase, as in Khairnar et al, 2003).
• Mox had seemed like an obvious choice because it would be sourced from Xanthobacter autotrophicus GJ10, the most well-documented DCA-degrader ([http://mic.sgmjournals.org/content/133/1/85.full.pdf).| Janssen et al, 1987])

aldA, chloroacetaldehyde dehydrogenase, dhlB, haloacid dehalogenase, and dhlA, haloalkane dehalogenase

• We used a sequence for aldA from Xanthobacter autrophicus GJ10, but used sequences for dhlB and dhlA from a different strain, Xanthobacter autrophicus EL4, which was isolated and characterised in the Coleman lab. When our Gibson Assembly ran into problems, we continued to work with dhlB and dhlA from EL4 in our lab and submitted these two genes as parts.
• These genes are shared by a few different species of bacteria that degrade DCA (Janssen et al, 1994), and most have been conventionally characterised by extraction and heterologous expression of a single gene at a time.

References

Khairnar, N. P., Misra, H. S., & Apte, S. K. (2003). Pyrroloquinoline–quinone synthesized in< i> Escherichia coli</i> by pyrroloquinoline–quinone synthase of< i> Deinococcus radiodurans</i> plays a role beyond mineral phosphate solubilization. Biochemical and biophysical research communications, 312(2), 303-308.