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Our project is in the track of Foundation Advancement; which is focused on providing technologies or parts that can benefit the sphere of synthetic biology or specifically the iGEM community and future competitors. With this in mind, our project's aim to is provide a technique that allows cells to have the ability to be localised to designated areas through genetic modification. Whilst in our project we are using E.coli as our model organism, the parts we create will be open to be trialled in other organisms. Researchers in microbiology and other fields can benefit from this work with the goal of being able to bring together cells of different types into contact. We are wanting to look more in depth into specific situations where this project can play an impact and be used. We are also looking to continually mould and adapt our work so any critique or questions that this work prompts we are very willing to hear about too.
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There are two parts to achieve our goal of precision control over the spatial arrangement of cells.
1) A new device that enables cells which can ‘stick’ and be directed to an area
2) A target to which the cells can then attach to.
Constructing cells with the ability to recognise and attach to a target is achievable in the realm of synthetic biology; by modifying a cell to express and display zinc finger proteins on the outer surface membrane. The target to which the zinc finger binds is to a sequence of DNA. The DNA will be arranged in a shape, as inspired by concepts from DNA origami, to fold into a planar shape with long extending arms. The end of the DNA arms will contain thee target sequences, to which the cells can adhere to. The arms of DNA origami structure can be designed and programmed to contain a variety of target regions therefore a combination of cells with differing zinc fingers can be brought together in an area seen in Fig.1.
The inspiration from this project has come from a paper on ‘barcoding cells’ by Mali et al. 2013 which achieved the display of synthetic zinc fingers on the surface of mammalian cells. This work was concerned with separating cells for cell labelling, tracking or for diagnostic purposes. We are adapting this idea by taking the perspective of bringing cells together rather than separating cells with the introduction of DNA structures that can hold together cells.
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Zinc finger proteins are intracellular molecules which recognise and bind unique dsDNA sequences. We have chosen a set of zinc fingers; 16 synthetic and already characterised from previous work, alongside several others such as Zif268. The mechanism of transporting the zinc finger to the outer membrane is through constructing a fusion protein with an anchor protein, which will be transported and embedded within the membrane. For certainty of getting a zinc finger to the membrane we have chosen 4 candidate anchor proteins to test, based on well documented research and whether some of which had been used with success by previous iGEM teams. From the collection of candidate anchor proteins and zinc fingers we aim to optimise this system, and have a collection of zinc fingers which allow multiple cells to be brought into contact.
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As a starting point we are conducting tests for the expression of the fusion protein, and working on a method of placing DNA on glass slides. Then we can test the binding of DNA to the slides in simple patterns to view the system. Currently we have demonstrated we can adhere oligonucleotides to the glass slide as shown in Fig.2. Fluorescent oligonucleotides (with amine groups) were introduced to 2 glass slides prepared; one treated with GOPTS, the other only cleaned. Both slides were washed and inspected with a FITC green filter. In theory only the treated slide should show fluorescence, and the results were as expected.
To demonstrate that the E.coli receiving our part construct are being expressed we added a flag tag to the part inserted, allowing us to treat cells with an anti-FLAG antibody and visualise using fluorescent microscopy. Wild-type, transformed and uninduced, and transformed and induced DH5α Z1 cells were dried onto cover slips and fixed. The cells were then treated with an anti-FLAG antibody, followed by an anti-mouse fluorescent antibody. The slides were then examined with a FITC green filter shown in Fig.3. Although random flourescence is seen in A this is likely due to poor antibody removal based on the areas of flourescence. The brightest floursecnce and clear cellular outlines are seen in C in the induced DH5α Z1 cells.
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A working model of how the DNA shape will be produced is described in Fig.4. We are currently collaborating with the Oxford iGEM team to produce this DNA structure.