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Team:NRP-UEA-Norwich/Project
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Please choose the relevant link to view an overview of each project!
Nitric Oxide Sensing & The Hybrid Promoters | The Comparator Circuit | Theoretical Projects
Sensory BioBrick systems have been a large constituent of previous iGEM projects in which teams have combined impressive amounts of logic with limitless creativity in order to produce synthetically engineered organisms with the ability to detect the presence of specific substrates; this was achieved by combining various promoters and reporters to produce novel gene systems of great breadth and depth.
We too have taken a sensory approach to our project and have produced systems involved in the sensation of nitric oxide (NO). Originally we set out to develop a bacterial and mammalian hybrid NO-sensing promoter (which we have achieved); we then looked into ways of quantifying the levels of highly reactive and difficult to measure NO within a system, leading to us producing a novel gene regulation system known as the comparator circuit. Throughout the project we went on to look at theoretical alternative approaches to the gene systems we have produced.
Overall in our project we have produced 6 sensory BioBricks, 2 BioBricks involved in gene regulation, and have further characterised 4 more BioBricks. All 8 of our BioBricks have been submitted to the registry and the 6 sensory BioBricks have been characterised.
Our own hybrid promoter hopes to add to the systems already in the registry by creating a hybrid promoter that combines the bacterial promoter PyeaR and the mammalian CArG element , both of which respond to exogenous nitrogenous species. Combining the two would allow a more modular NO sensor that can be used in mammalian and bacterial cells interchangeably.
The University of East Anglia has a great deal of research groups studying nitrogenous species, therefore we decided to look into using synthetic biology to try and help solve any problems they had experienced; one of the main issues was with the use of nitric oxide, as it is difficult to work with due to its highly reactive nature. We decided to take on this challenge and investigate how we could use synthetic biology to quantitatively measure NO levels in an environment.
We began by synthesising a dual promoter taking elements of bacterial NO-sensing promoters and mammalian NO-sensing promoters; this has led to our hybrid promoter, a combination of the PyeaR promoter (bacterial, given as 'B' in our project) and the CArG promoter (mammalian, given as 'M' in our project). These two elements have been synthesised in two orientations; mammalian upstream of bacterial (M-B) (Figure 1) and bacterial upstream of mammalian (B-M) (Figure 2); this allows our promoter to be flexible and have use in both prokarayotes and eukarayotes.
Over the course of our project we have produced six individual BioBricks using these hybrid promoters; M-B alone, M-B with RFP and M-B with CFP; and B-M alone, B-M with RFP and B-M with CFP. The BioBricks containing fluorescent proteins were transformed into an Escherichia Coli chassis and through induction by potassium nitrate (using the nitrate as a homogenous substitute for nitric oxide) we have observed expression of RFP and CFP. We have also conducted experiments to measure the level of fluorescence (and thus transcription) under different concentrations of potassium nitrate; to do this we carried out flow cytometry, fluorescence-activated cell sorting, and scanned samples using a fluorometer. Our data thus far has suggested that in the case of CFP, M-B has a higher transcription rate than B-M; however, in the case of RFP both M-B and B-M appear to have similar rates of transcription. In the majority of fluorometer experiments it appeared that the maximum concentration of potassium nitrate that the cell could cope with was between 15 and 20 mM, as 15 mM gave the highest fluorescence intensity, while 20 mM showed much lower fluorescence intensity; we hypothesise that this is due to 20 mM potassium nitrate being toxic, or due to over-expression of the fluorescent proteins resulting in aggregation and the formation of inclusion bodies.
We have also transfected mammalian cells with M-B attached to CFP and added SNAP (a nitricoxide-producing agent) in order to induce transcription. Results appeared to show low levels of CFP expression in cells without SNAP (pertaining to normal physiological levels of nitric oxide) and higher levels of CFP expression once SNAP had been added.
The flexibility of the hybrid mammalian and bacterial dual promoter allows for many advances in sensation and control of nitric oxide. For instance NO has been recognised for its implications as a potential cancer therapeutic (due to macrophage usage of NO as an immune response) therefore this promoters allows for the development of systems in easier to use, faster-growing bacteria that can then be applied to mammalian cells for testing. We believe this to be a novel approach made possible through our BioBricks.
The lack of specificity of the bacterial promoter, pYEAR, used in the hybrid promoter was a pitfall that was always a concern. From this potential problem spawned a potential solution; the Comparator Circuit.This pair of BioBricks is designed to specifically bind to each other while ligated to two different promoters of overlapping specificity to result in an integrating of the conflicting outputs of the two opposing gene systems.
Our system relies on two constructs that interact via complimentary base pair sequences both before and after the ribosome binding site of the reporter protein. The idea being that, when both transcripts are present in the chassis, they would bind together, inhibiting the translation of the reporter proteins.
Any imbalance of transcription due to the presence of the substrate of interest results in free mRNA of the gene system that detects that substrate. Crucially, if both promoters detect the same substrates but differ with one extra substrate being detected by one of the promoters, it is this substrate and this substrate only that our system will be able to detect in a simple and quantitative way.
Our team have constructed a countercurrent comparator circuit in which the reporter proteins are at the same end of the complimentary region, although a contracurrent system has been theorised. Both systems share a crucial subtractive nature comparable to an analogue computer. We envisage that, should the system be fine-tuned and expanded on, a variety of different business sectors from agriculture to spinoff pharmaceutical companies (Please check out our Quanticare page for more details
) could capitalise on this novel genetic technology.
What we have produced is a biobrick pair that work in harmony, when ligated to promoters of interest and genes of interest, to sequester translation when both mRNA transcripts are present in the cell. The use of quantative tuners with these biobricks is encouraged to ensure that the transcription rate both gene constructs are equal when both promoters are transcribing at their optimal rate. Although the parts have been submitted to the registry and theoretically characterised, time constraints have meant that further lab-based characterisation could not occur.
However, we hope to utilise any free time in our timetables sduring the next semester to characterise the biobricks further (please see our project proposal), and hope that we will be given a chance to present our further findings at MIT!
To conclude, what we have created is a pair of antagonistic BioBricks that turned the pair of mRNAs in which they reside into translational repressor molecules when both are transcribed in tandum within a specific chassis of interest, a new application for mRNA complimentary base pairing within the registry and a project we feel could go very far indeed.
Division circuit
The comparator circuit that we have created integrates two different transcription levels in a negative manner (subtraction) to perform a range of functions a range of different integrations (calculations) are necessary to this end we have also designed a system that would allow one signal (transcription rate) to be divided by the transcription level of another promoter. To achieve this we have used the processes of attenuation and the three loop system (in the tryp leader). the system has also been mathematically modelled. to find more please click the image above.
Multisensor
There are many groups of chemical species for which there are no current biological techniques for distinguishing between each of these species and quantitatively analysing its concentration. Here we outline a possible approach for solving this problem using non-specific transcription factors and promoters. We use nitrates nitrites and nitric oxide as our example group. To find more please click the image above.
in addition to the two main projects, we have also worked to elaborate on some of the teams earlier ideas during the project.
