Team:METU Turkey/Project

 

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.

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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.

Background

Carbon monoxide (CO), one of the most harmful pollutants, presents a significant effect on human health. It interacts with the hemoglobin protein of erythrocytes upon inhalation The oxygen attached to the hemoglobin is displaced with CO, and carboxyhemoglobin (COHb) is formed. CO’s affinity to hemoglobin is higher than that of oxygen’s. This strong COHb bond makes it difficult for the body to eliminate CO from the blood. Therefore, tissues and organs will suffer from oxygen starvation which can cause severe damage and even death.

The degree of injury inflicted by CO is proportional to the exposure concentration. Although it would take some time for low levels of carbon monoxide to show any effect, high levels can kill in less than five minutes.

Since carbon monoxide has no distinct color or smell, detection of this gas is actually quite a challenge. There are various types of detectors in the market that employ different methods of sensing. While some of them are designed to discern CO presence chemically or electrochemically, others use semiconducting and spectroscopic properties. A detector should be able to detect a concentration higher than 100 ppm and sound an alarm as a warning.

Soil, sediment, and water toxicity can be displayed as detectable and quantitative signals by the introduction of transducers to the bacteria (Biran et al., 2003). These bacterial biosensors contain a reporter gene that produces a signal and a contaminant-sensing gene that responds to changes of condition in the environment, such as exposure to a specific analyte. Such a change would cause the activation of the sensing component, which consequently activates the reporter gene through biochemical reactions. (Biran et al., 2003; Tauriainen et al., 2000, Turpeinen et al., 2003; Daunert et al., 2000). A gas sensor can be manufactured by the utilization of heme-containing molecules since it has an active site where it can attach to the gas molecules. (Aono et al., 2003).

One microorganism that can be used as a biosensor is called Rhodospirillum rubrum which is able to use CO as an carbon source with its CO-sensing protein called CooA. CooA activates the transcription of genes encoding the CO-oxidation (coo) regulon which produces the proteins needed for the utilization of CO.

The superfamily of transcription factors as cAMP receptor protein like (CRP) and fumarate and nitrate reductase regulatory protein like (FNR) comprises the proteins that respond to wide range spectrum of intracellular and exogenous signals in a direct or indirect manner. CooA is the member of this family (CRP/FNR) as single component regulatory proteins. It is reported that CooA includes a b-type heme that in vivo conditions acting like CO sensor. Carbon monoxide can bind to the ferrous heme group of CooA. CO bound CooA undergoes a conformational change and becomes active as transcriptional activator that binds to DNA as sequence-specific.In absence of carbon monoxide,protein can not bind to DNA. Active CooA binds to DNA in a position overlapping the -35 element of the P(cooF) promoter.

Figure 1. Crystal structure of CooA.

The two CO regulated transcriptional units, cooMKLXUH and cooFSCTJ, is responsible for the CO-dependent anaerobic growth of Rhodospirillum rubrum and enable the bacteria to oxidize CO. CooA is responsible for the expression of the genes of these proteins and activates by sensing CO in anaerobic environments. The coo regulon has three main products which are listed as O2-sensitive CO dehydrogenase (CooS), CooS-associated Fe-S protein (CooF), CO-tolerant hydrogenase (CooH).

PcooF and PcooM promoters have 2-fold symmetric DNA sequences that resemble CooA- binding sites, similar to CRP consensus sequence. Coinciding with 235 region, the CooA binding sites lie at the 243.5 and 238.5 locations relative to the transcription start sites in PcooF and PcooM. This overlapping reveals that PcooF and PcooM are similar to the class II CRP-dependent promoters (He et. al., 99). We prefer using PcooF as it is stronger than PcooM, depending on the primer extension product amount and level of coo-encoded proteins synthesized in vivo. Because of the lack of active CooA, CO-independent basal level transcription of PcooF is not detectable.

References:

1) Shelver D, Kerby R.L., He Y, and Roberts G.P. (1997). CooA, a CO-sensing transcription factor from Rhodospirillum rubrum, is a CO-binding heme protein. Proceedings of the National Academy of Sciences 1997; 94: 11216-20.

2) Aono S, Matsuo T., Shimono t., Ohkubo K, Takasaki H. and Nakajima H. (1997). Single Transduction in the Transcriptional Activator CooA Containing a Heme-Based CO Sensor: Isolation of a Dominant Positive Mutant Which Is Active as the Transcriptional Activator Even in the Absence of CO. Biochemical and Biophysical Research Communications 1997 Oct 7; 240,783-786.

3) Aono S (2003). Biochemical and Biophysical Properties of the CO-Sensing Transcriptional Activator CooA. Accounts of Chemical Research 2003; 36(11): 825-831.

4) He Y, Gaal T, Karls R, Donohue T, Gourse R, and Roberts G (1999). Transcription activation by CooA, the CO-sensing factor from Rhodospirillum rubrum. The interaction between CooA and the C-terminal domain of the alpha subunit of RNA polymerase. The Journal of Biological Chemistry 1999 Apr 16; 274(16): 10840-5.

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