Team:SydneyUni Australia/Project/Parts

From 2013.igem.org

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__NOTOC__
== '''Project Registry Parts'''==
== '''Project Registry Parts'''==
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<center>
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{|class="wikitable sortable"
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! !!  !!  !! Name !! Type !! Description !! Designer!! Length
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== '''Gibson'''==
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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. 
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* 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.
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|[http://parts.igem.org/Part:BBa_K1115004 <span style="color:red;">BBa_K1115004</span>]
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* 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 [https://www.idtdna.com/pages/support/technical-vault/webinars/categories/synthetic-genes/rapid-and-reliable-gene-construction| webinar].
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| Transcriptional Unit
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* 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].
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| dhlA Haloalkane dehalogenase
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| Sydney University
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== '''Design'''==
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| 958
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=== '''Choice of genes'''===
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'''Mox, chloroethanol dehydrogenase'''
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|[http://parts.igem.org/Part:BBa_K1115007 <span style="color:red;">BBa_K1115007</span>]
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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).  
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| Transcriptional Unit
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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])
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| dhlB 2-haloalkanoic acid dehalogenase 
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| Sydney University
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| 791
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|[[File:heart13.gif]]
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|[http://parts.igem.org/Part:BBa_K1115008 <span style="color:red;">BBa_K1115008</span>]
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| Transcriptional Unit
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| dhlB + dhlA ligated with HindIII 
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| Sydney University
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| 1744
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|-
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|[[File:heart13.gif]]
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|[http://parts.igem.org/Part:BBa_K1115009 <span style="color:red;">BBa_K1115009</span>]
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| Generator
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| Constitutive production (PCat) of dhlB and dhlA
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| Sydney University
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| 1790
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|-
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|[[File:heart13.gif]]
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|
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|[http://parts.igem.org/Part:BBa_K1115010 <span style="color:red;">BBa_K1115010</span>]
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| Generator
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| Constitutive production (PTet) of dhlB and dhlA
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| Sydney University
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| 1806
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|}
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</center>
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== '''References'''==
 
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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.
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{{Team:SydneyUni_Australia/Footer}}

Latest revision as of 23:51, 27 September 2013

SydneyUniversity Top Banner.jpg SydneyUniversity Bottom Banner.jpg


Project Registry Parts

Name Type Description Designer Length
BBa_K1115004 Transcriptional Unit dhlA Haloalkane dehalogenase Sydney University 958
BBa_K1115007 Transcriptional Unit dhlB 2-haloalkanoic acid dehalogenase Sydney University 791
File:Heart13.gif BBa_K1115008 Transcriptional Unit dhlB + dhlA ligated with HindIII Sydney University 1744
File:Heart13.gif BBa_K1115009 Generator Constitutive production (PCat) of dhlB and dhlA Sydney University 1790
File:Heart13.gif BBa_K1115010 Generator Constitutive production (PTet) of dhlB and dhlA Sydney University 1806


With thanks to: