Team:METU Turkey/Project

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ABSTRACT

Cells can sense and respond to the presence of various gas molecules such as oxygen, nitrogen and carbon monoxide using gas sensor proteins.

CooA is a carbon monoxide (CO) sensing transcription factor. It is a member of the cAMP receptor protein (CRP)/fumavate nitrate reduction (FNR) family of transcriptional regulators. CooA switches on oxidation enzymes in Rhodospirillum rubrum (a purple, nonsulfur, phototrophic bacterium) which enables the bacterium to use CO as a carbon source.

CO is an odorless and colorless gas which can be extremely lethal. Our aim is to develop a cell sensor which can detect a wide range of CO concentration in the environment.

We are building CooA and CooA-responsive promoter biobricks which will be transformed into E.coli. Fluorescent proteins (GFP and RFP) will be utilized as dose-responsive signals of ambient CO.

PROJECT DESCRIPTION

Objectives

- To construct a carbon monoxide sensing cell sensor
- To increase the dynamic range of CO sensor with strong/weak response element coupling

Enhanced Dynamic Range (EDR)

In a typical biphasic binding event in which strong and weak affinity binding interactions can take place between two molecules, saturation of the strong affinity site is followed by a second saturation event of the weak site. Based on this principle, we coupled strong and weak binding response elements of carbon monoxide sensing transcription factor CooA to two different signals, GFP and RFP respectively. In this way, we expect not only to detect the presence of carbon monoxide gas but also increase the dynamic range of our cell sensor using strong-weak promoter coupling.

One of the important questions we are hoping to answer is how many fold affinity difference between CooA and its response elements (RE) would be required to so that when we couple these REs we obtain the widest possible dynamic range for the CO detection?

How E-CO Sensor Works?

When CO is introduced into the medium, transcription from both strong and weak CooA responsive promoters will be initiated. Since affinity of CO bound transcription factor is higher for the strong promoter, GFP signal will dominate the RFP signal due to the higher transcription rate of the former. Increase in CO concentration will completely saturate strong promoter and after a point saturation of the second, weaker promoter will begin. As the concentration of the signal from weak promoter (RFP) increases, detected fluorescent signal will start to change from green to yellow.

Components

- pCooF+rbs+GFP+tt – This component is used to measure low levels of carbon monoxide in the environment. Construction of this device was performed by utilizing strong CO sensitive promoter which is pCooF promoter from Rhodospirillum rubrum. This device is only active when there is CooA transcriptional activator and Carbon monoxide gas in the environment. When there is CO in the environment, the device produces green fluorescent protein in response.

- pCooM+rbs+RFP+tt – This component is used to measure high levels of carbon monoxide in the environment. Construction of this device was performed by utilizing strong CO sensitive promoter which is pCooM promoter from Rhodospirillum rubrum. This device is only active when there is CooA transcriptional activator and Carbon monoxide gas in the environment. When there is CO in the environment, the device produces red fluorescent protein in response.

- pTriEx +CooA – This component is used for transcriptional activation of pCooM and pCooF promoter. When CO is present in the environment, CO binds to heme group of CooA protein which opens the binding sites of CooA protein. The CooA gene is ligated into the pTriEx vector which expresses CooA protein in high amounts. This component is used to detect CO presence. It expresses RFP in low concentration when there isn’t CO present. When there is CO, CO binds to CooA and expression increases substantially.

- Final Construct – This construct has been done by ligation of pCooF and pCooM promoters which are coupled to GFP and RFP proteins. It is designed as CO sensor whose data gives an approximate data about CO amount in the environment -

Execution

Our research group were divided into four teams to design and characterize E-CO Sensor. Alpha Team is responsible for cloning and cell sensor experiments; CooA overexpression and purification will be performed by Bravo Team; Charlie Team will mainly contribute with their expertise in fluorescence spectroscopy and confocal laser scanning microscopy; Delta Team will be performing characterization experiments and will be joined by Bravo when protein expression/purification package is completed.

The project will start with the design and ordering of CooA (wild type/mutants) and its response element (wild type/mutants) sequences. CooA mutants were reported to have higher affinity for CO. They were included in our order to shorten CO response time if needed. Two promoters, PCOOF and PCOOM, were previously reported as strong and weak promoters of CooA. We will also design several PCOOF promoter mutants (point mutations). These mutants are expected to have changed affinity for CooA. Binding affinity of CooA and mutated promoters will be determined as a part of characterization work package which includes Isothermal Titration Calorimetry (ITC), Electrophoretic Mobility Shift Assay (EMSA) and Intrinsic Tryptophan Fluorescence (ITF). Following this, promoters with different CooA affinities will be coupled and constructs will be prepared for in-vivo cell sensor experiments.

In cloning package various strong/weak promoter couples followed by GFP and RFP signals will be produced and co-transformed with CooA vector into E.coli. Cell sensor experiments will proceed in three stages; Fermentor, flask and anaerobic chamber (solid culture) experiments. In all these, cell sensors will be tested under various culturing conditions including IPTG induction concentration and time. Growth curves will be constructed and CO will be introduced at different growth points and at varying concentrations. Dissolved CO will be determined using a myoglobin assay which is based on the absorbance change of myoglobin upon binding to CO. Cell response (GFP/RFP signals) will be measured by confocal laser scanning microscopy and fluorescence spectroscopy.

We will compare and correlate our findings from in-vitro binding experiments and in-vivo cell sensor studies to develop an optimized CO cell sensor with enhanced dynamic range.