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Future Work
SherlocoliMainly, Sherlocoli aims to detect the tumor cell in the media via the binding reaction occurring between CD215 antibody and EpCAM. We have proofed successfully that our binding docks are fully synthesized and, most probably, are able to bind EpCAM strictly. This makes us think that the detection of CTCs as a result of binding to an EpCAM antigen with C215 antibody is actually possible. The reporter signaling pathway should be activated in a way; thus, the next thing we are going to investigate is how we can proof the transfection reaction occurring in the bacteria.
As a result of Co-Immunoprecipitation, treating the lysate with anti-flag antibodies and SDS-Page imaging, we were able to show the right proteins and protein junctions. Herewith, we can show easily that the FLAG-peptides are expressed. What we plan to do next is to reproof our system with Western Blotting so we can isolate only our desired proteins completely.
We plan to add an appropriated signaling pathway at the end of the module; so, we can intensify our experiment separately from the LasR induction on the optimization.
Using microorganisms for the detection at a microscopic level could find many application areas in the future. Especially in the medical sector, there could be many possibilities for detecting various disease markers after complete optimization of the bacterial detection system for the clinical use and the specific markers are discovered. It should be considered that the current detection techniques are expensive and time-consuming; but, a modified organism with fast response ability may reduce the risks that occur because of the time that is lost unnecessarily.
Only disadvantage that our project poses is the presence of EpCAM on normal tissue cells. EpCAM is synthesized at high amounts in the tumor cells, which is well demonstrated in the literature; but fibroblasts and lymphocytes are also able to produce such physiological receptors. The thing that our project offers here is a proof of concept which gives promising research field for the scientists that a modified organism can be used as tumor detector without threatening any safety issues. In our model, CD215 is effectively used to indicate EpCAM antigen; thus, in the course of well development in next few years when some new cancer markers and targets would be identified, these model organisms can be modified with specific antibodies which may pose a promising role in the diagnosis of cancer.
DJcoli
In this module, we plan to use the quorum sensing mechanism active as a signal amplifier and a synergic response generator. So far, we could not manage to clone our quorum sensing devices correctly; but the signal inducer anchor-inducer proteins are confirmed in gel electrophoresis. We want to finish our cloning session as a next step and, hopefully, to start the assays on this mechanism.
As the first experiment, we plan to induce a bacteria colony with the diffusion of quorum sensing chemicals, AHLs, which are intended to be synthesized by another colony in the same media. The reporter protein that is produced by the receiver colony will indicate whether our model is working properly.
Aftermath, we want to test the anchor-inducer protein complexes in order to investigate the relation and correlation between the detection signal and the amplifier. To do this, we plan to add our anchor-inducer protein sequences just after the detection device. (BBa_K772100) Another colony will be modified with receiver device in order to measure its reaction to the transfection occurred in the sender cells.
In the future, the quorum sensing effect may be used as alternative modification technique instead of BioBrick Assembly Standards. Because of the toxicity or the huge length of plasmids with modified genes, alternative methods have gain absolute importance. In the model of quorum sensing device, it is needed to modify at least two different colonies with different genes; but its success rate seems higher. Because of this reason, any contribution to improve this system is also important. In our project, we propose an alternative device mechanism to induce quorum sensing.
Quorum sensing also offers a method that amplifies its original yield. This may lead the generation of sensitive detection systems in the water distribution canals or public spaces. This may prevent the possible healthcare problems or the exploration of unknown information.
Dracoli
Basically, Dracoli module focuses on how we can reduce the risk of the production of possible GMOs (Genetically Modified Organism) in the media. To do this, the module uses chloride ion transfer into the cell which ends with the induction of an inducible promoter that the whole process is activated with the emission of light. This allows us to tune the whole system with different parameters. (Wavelength of light, exposure time, chloride gradient etc.)
So far, some parts of this module are assembled together and ready-to-use for the main goal: to induce self-destruction after seeing a particular light. (BBa_K772101, BBa_K772102) Our next step will be to characterize these parts and add some more subparts in the downstream region. Finally, we hopefully want to demonstrate that our bacteria can actually find cancer cells and can kill themselves if it is necessary.
Halorhodopsin, chloride sensing device and our LALF need further investigation about their abilities. A chloride transfer assay would hopefully give us information about the characterization of halorhodopsin and chloride sensing unit. Also, we want to carry out further investigations about the BioBrick of last year, LALF, in order to stop the bacterial growth.
In the next years, tunable reporter systems will become more important. So far in iGEM, the reporter devices give opinion for only the existence of the fact. The level of that factor would not be well-documented or to demonstrate such conditions may become difficult. The tunable systems solve these problems and also proposes easy regulation for simple devices.
Tuning the self-destruction is also promising for the future. This can be used to control the volume of the population. In our model, this tuning ability is sourced by light which is very cheap, easy-to-get, easy-to-use and effective. This is one of the reasons that light driven ion pumps and their inducible promoters are one of the most important subparts for the future projects.
