IGEM:Hong Kong HKUST/Investigations/Investigation on the Light-Sensitive amilCP Generator

Introduction 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. Method The construct was constructed by digestion and ligation method.BBa_K592016 was digested and extracted as insert and it was ligated to BBa_J23101 which act as vector. Then these two parts were putting into plasmid containing BBa_K592020. The negative control was cells with BBa_K592020 without BBa_J23101 and BBa_K592016.The reason for that was to ensure that sensor part BBa_K592016 was needed for change in presence or absence of light.It was expected that if there is no sensor part,it must give negative result.The construct was tested by preparing two LB-agar plates(X,Y) which halves of each with colony having the construct and negative control colony.Then,plate X was wrapped with Aluminium(Al) foil to provide dark condition.Both plates were incubated overnight under light exposure. Result and interpretation An assumption was made which is the heating effect of light was negligible.In plate Y(light)colony with construct showed pale blue because light repress FixJ activation so λcl(repressor) transcription was inhibited.As a reult,amilCP transcription was allowed which led to production of blue color.But in plate X(dark)colony with construct showed white beacuse when there is no light,FixJ activation was not repressed.λc(repressor) transcription was activated,thus amilCP transcription was inhibited.Therefore,there is no production of blue color.In both plate, negative control colony showed deep blue.It may because there is no part B,so promoter pFixK2 was not activated.Therefore,constitutive production of amilCP cause deep blue color.By comparing the color change in colony with constrcut under presence and absence of light,it shows that sensor part in BBa_K592016 is needed for color change. Discussion 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. Conclusion The biological sensor constructed could differentiate presence of light with response by color production Attribution Authors:CHEUNG Ming Fung ,Leung Pok Man,Chan Tat Yin Web page created by Yip Chun Yin