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) and bacterial upstream of mammalian (B-M); this allows our promoter to be flexible and have use in both prokarayotes and eukarayotes.
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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.
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 alternate 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.
Figure 1. A graphical representation of the hybrid promoter in its Mammalian-Bacterial orientation, also showing restriction sites.
Figure 2. A graphical representation of the hybrid promoter in its Bacterial-Mammalian orientation, also showing restriction sites.
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.
Following the ten weeks 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 in the relevant systems. 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 as shown that CONCLUSIONS.
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 are designed to specifically bind to each other while ligated to promoters of overlapping specificity to result in an integrating of the conflicting outputs of the two opposing gene systems.
In essence, 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.
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