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From 2012.igem.org

Overall project

Abstract: Phytochromes are used in organisms to sense light and control the expression of genes, bacteria specifically use bacteriophytochromes. A bioswitch will be created using the phytochromes from Deinococcus radiodurans and Agrobacterium tumefaciens. Through the technique of Gibson Assembly, the coupling of heme oxygenase supplies our phytochrome proteins with biliverdin, allowing for the self-assembly of the switch within host systems. The completed bioswitch will demonstrate a noticeable colour change from blue to green when exposed to red and far red light respectively. This when coupled with an appropriate response regulator can enable the control of targeted gene with the input of light.

Bacteriophytochrome

Hemeoxygenase

Project Aims: The objective of this project is to therefore build and characterise a biological light switch in E. coli. This will involve construction of heme-oxygenase and bacteriophytochrome BioBrick parts. This year's research team will be expanding upon the research conducted by last year's iGEM team and the team from 2010. In 2010 the Macquarie Team cloned bacteriophytochrome from two sources. They showed that when one was expressed, it was functionally assembled when incubated with exogenous biliverdin and able to elicit a colour change when excited with far-red light. However, the part created is not directly usable as a BioBrick as it contains an internal EcoRI site (Deinococcus radiodurans phytochrome) and 2 PstI sites (Agrobacterium tumefaciens phytochrome). As biliverdin is not native to E. coli, the addition of heme oxygenase is required for the synthesis of bilivedin, enabling the self-assembly of the light switch. In 2011, the Macquarie Team successfully managed to construct and characterise the heme oxygenase 1 as a BioBrick. They showed, via its green color, that cells expressing the heme oxygenase could degrade heme into biliverdin.

In this project we will aim to construct the BioBricks using Gibson cloning as opposed to restriction enzyme digests.

Project Work Flow

1. Design of Heme oxygenase and two bacteriophytochrome fragments
2. Approval and synthesis of fragments by Integrated DNA Technologies (IDT)
3. Assembly of fragment sequences into BioBricks using Gibson Assembly
4. Transform into E. coli
5. Sequence
6. Assemble BioBricks according to NEB & Gingko Bioworks Protocol
7. Transform into E. coli
8. Test for function and sequence (26th September: Parts must be sent to registry by this date)

Results