Part:BBa_K1954001:Design
Lycopene cassette under the control of NarK, an oxidative stress inducible promoter
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 2121
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1657
Illegal NgoMIV site found at 1787
Illegal AgeI site found at 872 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
We combined the lycopene BioBrick already existing in the registry with the NarK promoter in order to control the expression of lycopene. The BioBrick falls into two parts:
Sensing The promoter senses oxidative stress and in response turns on transcription of the downstream genes. Responding The gene for the antioxidant, lycopene is turned on as a response to oxidative stress.
Lycopene-NarK initial design:
Further steps were taken in the design process:
1. Removal of illegal restriction sites within the gene through silent mutations. 2. In order to minimise the time to receive DNA from IDT, design steps were taken in order to put these into gBlock format. This required splitting up of the rather large gene construct into 4 parts of around 500 bp as well as consideration for the collating of the parts into one by Infusion.
The final outcome was a composite BioBrick, BBa_K1954001, which we submitted to the registry.
We wanted to find validated protocols for the expression and detection of lycopene. Firstly we wanted to induce oxidative stress. After several options were considered, from nitrogen gas to hydrogen peroxide we went with a protocol involving nitrate, nitrite and NO. Secondly we wanted to measure lycopene. We adapted the protocol used by Cambridge 2009 iGEM as they had success and produced data, suggesting we would also be able to follow this.
The detection of Lycopene is simple since it has a distinctive red colour enabling it to be determined by spectrophotometry.
Source
TO BE UPDATED