Team:Macquarie/Protocols/Designing Gibson Assembly Fragments

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Revision as of 02:16, 21 August 2012

Haemoxygenase

Original Gene Sequence: http://www.ncbi.nlm.nih.gov/nuccore/BA000022

Team mates performing codon optimisation

Deinococcus radiodurans Bacteriophytochrome

Original Gene Sequence: http://www.ncbi.nlm.nih.gov/nuccore/15807672/?from=53908&to=56175&strand=true&report=genbank

Agrobacterium tumefaciens Bacteriophytochrome

Original Gene Sequence: http://www.ncbi.nlm.nih.gov/nuccore/159184118/?from=1954292&to=1956502&strand=true&report=genbank

BioBrick assembly using Gibson method: Fragment design

• Codon optimisation:

The Amino acid sequences for Haemoxygenase (synechocystis) and Bacteriophytochrome (Deinococcus radiodurans and Agrobacterium tumefaciens) were initially run through the Reverse Translate program (http://www.bioinformatics.org/sms2/rev_trans.html) for codon optimisation in E. coli BL21 .

• BioBrick standardisation:

Each sequence must conform to standard BioBrick part specifications which can be found here (http://partsregistry.org/Help:BioBrick_Prefix_and_Suffix). These standard components were added to our sequences in silico with the addition of an appropriate promoter such as T7 (http://partsregistry.org/wiki/index.php?title=Part:BBa_I712074) (Required for E.coli BL21) and a RBS/Shine-dalgarno sequence which was taken from the iGem 2010 Macquarie team (http://partsregistry.org/wiki/index.php?title=Part:BBa_K646000).

Standard BioBrick Structure: EcoR1-Xba1-T7-RBS-ATG-(Gene insert)-TAATAA-Spel-Pst1.

Finally each DNA sequence was run through the restriction digest program (http://tools.neb.com/NEBcutter2/). This was performed to determine if the BioBricks contained any of the standard restriction sites such as EcoR1-Xba1 -Spel-Pst1 internally. These must be removed from the DNA sequences by changing codon usage in that sequence without changing the Amino acid sequence. We found that Agrobacterium tumefaciens and also Deinococcus radiodurans contained internal Pst1 restriction sites which we had to removed from the in silico sequences.

• Cloning gBlocks™ Gene Fragments using the Gibson Assembly™ Method:

Each BioBrick is going to be assembled using the Gibson Assembly™ Method using synthetic 500 bp fragments (gBlocks) that have 30bp even overhangs (http://www.idtdna.com/pages/products/genes/gblocks-gene-fragments) (http://www.idtdna.com/pages/docs/synthetic-biology/gblocks-user-guide.pdf).

First each Biobrick sequence was broken up into fragments of less than 500 bp (gBlocks). To these gBlocks overhangs were designed so that the 5' end of each fragment consisted of the 3' end of the flanking fragment. The starting 5' fragment of each given BioBrick has a 30bp overlap with the linear expression vector pSB1C3(http://partsregistry.org/wiki/index.php/Part:pSB1C3).

• Feedback and Issues Experienced:

Each gBlock fragment is synthetically produced in a process which is sensitive to GC rich regions which can introduce hairpin loops. GC% rich regions and hair pin loops were edited a number of times according to IDT feedback. Changes to these regions required adjustment of codon usage in to the next best E.coli codon without changing the amino acid (as determined by the percentage usage in native E. coli genes. These sequences then had to be double checked for restriction sites which may have been introduced by the CG content changes. On several occasions these edits included the addition of restriction sites which had to be removed by further editing.