Switch-a-roo: engineering a photoresponsive E.colight switch Team - PowerPoint PPT Presentation

Switch-a-roo: engineering a photoresponsive ‘ E.colight switch’ Team Macquarie University Sydney, Australia

What are the elements of ‘switch -a- roo’?  Phytochrome  Protein that binds chromophore  Chromophore PDB 1ZTU  Molecule that reacts to light  Light  660-800 nm range biliverdin

Mechanics of the Light Switch PDB 209C Biliverdin + ACTIVE Bacteriophytochrome INACTIVE HIGH ratio of light absorbed HIGH ratio of light absorbed in in far-red light range (730- red light range (660-730nm) 800nm)

What were our bacteriophytochromes?  Four domains  Agrobacterium tumefaciens  Able to transfer and integrate DNA into host genome  Deinococcus radiodurans  Extremely radiation PDB 209C resistant

Heme Oxygenase makes biliverdin  Biliverdin produced from the catabolism of heme by Heme Oxygenase 1  ALA ( δ -aminolevulinic acid) is a precursor in the biosynthetic pathway of heme biliverdin heme

What we planned to achieve  Assemble an on/off light switch mechanism  Consisting of a T7 promoter, HO and bacteriophytochrome parts.  Construct BioBricks:  Heme oxygenase  D. radiodurans - phytochrome  A.tumefacians – phytochrome  T7 promoter

Final construct

Starting materials for BioBrick construction  Used the materials from MQ 2010 effort Gene product Template RBS + HO-1 pET3a-HO1 RBS + A.tumefacians pGemT-easy phytochrome RBS + D.Radiodurans Amplified PCR product phytochrome

BioBrick construction using RFP BB- vector  RFP BB (BBa_J04450)  Red colony  Successful insert  White colony  Easy visual analysis

HO1-BB construction HO-PCR Ladder product  HO amplicon  ~800bp 0.5kb After BioBrick construction  Initial screen  Digest with Xba I 1. Uncut plasmid 2. RFP 3. Uncut plasmid without 1 2 3 4 insert 4. Xba I digest of plasmid

Our first BioBrick! Plasmid  Double digest of Eco RI and Spe I backbone  Shows correct orientation 2.2kb 2kb  Sequencing 1kb 0.8kb HO fragment

BioBrick Assembly of HO + T7 T7 HO  T7 promoters used:  BBa_I712074 from 2007 Ljubljana Team.  BBa_I719005 from 2007 Imperial Team

BiIiverdin! The HO works  Transform into BL21(DE3) E. Coli  Contains T7 RNA polymerase

How we improved a BioBrick  Our new HO BioBrick improves part BBa_K497005  Their part lacks a RBS  Added RBS & proved functional BBa_K646000

Successful PCR of phytochromes  Amplicon size  2.3kbp AT-Bph DR-Bph 2kbp 2kbp

Next steps for our light switch  Construction of the phytochrome BioBricks  Removal of internal restriction sites  Complete assembly of the light switch Next T7 HO1 Bph Switch-a-roo

How is this useful?  Switch on using red light  Response time  Regulation of gene expression by light  Blood detector (assuming cell free system)

 Add team photo Thanks for listening!