IGEM:Hong Kong HKUST/Investigations/Investigation on the Light-Sensitive amilCP Generator
In this project, we aim to construct a biological light sensor which could produce blue chromoprotein only under exposure of light, by assembling 3 parts -- BBa_J23101(A), BBa_K592016(B) and BBa_K592020(C) accordingly. BBa_J23101 is a consititutive promoter. BBa_K592016 codes for the blue light sensor protein YF1 with its response regulator FixJ. K592020 consists of a downstream signaling promoter of YF1/Fix(FixK2), an inverter cassette (cI and pLambda) and the blue chromoprotein reporter (amilCP).
In theory, under dark condition, auto-phosphorylation of YF1 is much favoured over the de-phosphorylation. The phosphorylated YF1 activates pFixK2 promoter and then the production of the inverter cI is allowed, which inhibits pLambda promoter activity. Ultimately, amilCP transcription is prohibited, implying no colour production. Under blue light illumination, the auto-phosphorylation of YF1 is inhibited while the de-phosphorylation is favoured. Hence, the pFixK2 promoter is inactivated and thus the subsequent expression of the inverter cI.Then, production of amilCP is allowed by pLambda activation, expressing blue colour.
Result and interpretation
The following are possible errors involved in the experiment.First, there were multiple failures when transformating ligated DNA. It might be due to insufficient time for transformation or competence cells used had low permeability to long DNA. Second, there was once failure of ligation,which might be due to the fact that large vector and insert size reduce relative concentration of sticky ends leading to poor ligation. Or it might also because of the low concentration of BBa_K592016, as linear pSB1C3 has similiar size to linear BBa_K592016 leading to poor separation after gel electrophoresis and imposing difficulties on subsequent cutting and extraction, causing huge loss after gel extraction. And additional time was used to re-digest BBa_K592016 to obtain higher concentration. Ultimately, as each experimental step was time-consuming, accumulaton of failures exceeded the expected time to finish the construct.
Improvements for this experiment is needed.First,the distance from plates to light source should be set equal and measured to check the effect of heating problem.Second,the experimental construct should be provided more time to express amilCP.Third,the intensity of light should be investigated to find out whether it will affect expression of amilCP or not.
Further investigations are needed.First,threshold intensity for observable change should be tested.Also,time reqiured to show response should be found out.Moreover,effect of different light wavelength on the functionality should be investigated.
The biological sensor constructed could differentiate presence of light with response by color production
Authors:CHEUNG Ming Fung ,Leung Pok Man,Chan Tat Yin
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