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Team:UCL PG/Project
From 2013.igem.org
Live had been on earth for 3.6 billion years. Over time living-beings evolved, merged, devoured and mutated. Cells are having ever massive networks and interactions that is far beyond foreseeable. And now we are only slowly discovering the secret of life. Through this sea of proteins, fluorescent and bioluminescent proteins are like light houses that allows scientist to hold on with faith. As the saying goes, "Seeing is believing". Therefore, we decided to dedicate our iGEM project to this wonderful technology.
We focus on producing a dual light sensitive circuit in order to evolve a red fluorescent protein, mKeima. At the same time we wish to establish a noble concept of "Evolution circuit" and associated high-throughput evolution protocols.
The architecture of dual light sensor revolves around the trick of overlapping fluorescent spectra. In this project, we improvised the overlapping of mKeima emission and Cph8 excitation wavelength. They are fused together, becoming Cph8-mKeima chimera to bring them to close proximity. Cph8 is a fusion protein which was firstly used for bacterial photography.It is able to relay signal to E.coli via phosphorylation of OmpR.
In order to turn the dual light sensitive chimera to full usability, optimization of the protein structure is necessary. To achieve that, there are generally two ways: Rational Design or Directed Evolution. Rational approach is both time consuming and challenging unless the target protein had been well studied. Alternatively, directed evolution is tedious but they always work and it is where most of the time significant "surprises" are discovered. In our case where we are optimizing a chimera, rational approach is beyond interpretation. Hence, we setup a journey to engineer an evolution circuit.
An elegant solution of it is to introduce a eGFP reporter with Omp promoter to respond to level of phosphorylated OmpR. Omp promoter capture the signal and output it as eGFP expression which correspond to the strength of response to blue or red light. In short, the better is the mutant, the greener is the cell. This eGFP brightness can then be detected by cell sorter rapidly during screening step to pick out the good mutants.
We had biobricked mKeima as expression cassette into pSB1C3. On the other hand, we also extracted eGFP [] and mRFP [] expression biobricks from iGEM 2013 distribution kit and helped to characterize their specs. Transformed bacterias were grown up and lysed. Then we characterized their excitation and emission spectrum on 96 well plates. Note the huge stoke shift of mKeima compared to eGFP and mRFP.
(Download Data in Excel here)
pCSTA promoter is repressed by presence of glucose. It is used as an indicator of glucose level in media, a very useful component in bioimaging. However, one potential hurdle is the ability of fluorescence to shine out of thick, dense culture media. Considering application in vivo or mammalian cell bioreactor, higher readout wavelength is more favored due to low attenuation of red light through living tissues. Higher wavelength light can pass through living tissue easily because most living tissues consist of blood and they are red which means they absorb lights of wave length below red light.
In this experiment, we wish to show that mKeima can be brighter, stable and more quantitatively informative than common fluorescent proteins such as eGFP and mRFP. pCSTA.mKeima, pCSTA.eGFP and pCSTA.mRFP are first amplified in tubes overnight. Then their OD600 is measured and their density is normalized with dilution. The normalized cultures are grown in 96 wells plate over a glucose gradient in LB media with antibiotics overnight in platereader at 37C. Kinetic data is obtained over 12 hours at 20 minute intervals.
(Download Data in Excel here)
Graphs above show the fluorescent readouts at the end point of experiment. For kinetics data, please refer to the original excel file for more detail.
In this experiment only 200uL (in each well) of living bacteria culture is measured. Hence, the fluorescent readouts are expected to be low. The purpose of this is to test the ability of each fluorescent protein to give out dependable quantitative data at tiny scale which is exactly the scale which most molecular experiments are done. pCSTA.mKeima displayed a clear and smooth trends of glucose dependency. Following that mRFP also showed relevant dependency but the data is more unstable. However, eGFP is found to be very inappropriate to indicate pCSTA's dependency on glucose concentration. Firstly, the graph shape is distorted. Secondly, it is believed that there is a high autofluorescence background from bacteria itself, seriously increased the fluorescent readouts to a false reading of 20 folds higher compared to mKeima and mRFP.
To understand the effect of attenuation of light across culture media or living tissue, we modeled a system based on Beer-Lambert law.
eGFP and mRFP have considerable spectral emission overlap and require post-acquisition compensation to distinguish them. Although eGFP and mKeima are both excited using the blue laser, there is considerable separation in their emission spectra allowing easy separation. Furthermore, mRFP and mKeima can be effectively distinguished from each other using different excitation lasers.
Work under progress ;D
Lab
Ethidium bromide (carcinogenic chemical) is used during gel electrophoresis to visualize the DNA. A designated EtBr work area is allocated in the lab for EtBr works only so that it does not contaminate other lab equipment possibly touched with bare hands. The EtBr liquid waste is incubated with green bag kit before disposal.
Machines like platereader and cell sorters are operated under supervision of trained personnel.
PPE is always worn in the lab without exception.
Environment
Lab waste containing cells are disposed in yellow bags and ultimately incinerated. The purpose is to ensure to antibiotics resistance bacteria from lab escape to wild nature.
We always recycle our pipette tip racks :D
