Difference between revisions of "Team:Warwick/Modelling3"

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<h4>DNA Origami Glue</h4>
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<h4>3D Printing Complex Structures using Secreting Cells</h4>
 
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<h5 class="sidebartitle">How it would work</h5>
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<p><img src="https://static.igem.org/mediawiki/2015/c/c4/Warwickbubbles8.png" height="120px" width="800px" border="1px"></p>
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Once we had an idea of how we could create small shapes using cells we wanted a quick overview how larger objects could be made. This discusses the different possibilities of the creation of 3D structures and shapes in a general sense, using different forms of bio-printng.
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<p>____________________________________________________________________________________________________________________________________________________</p><h5>The Problem</h5>
 
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<p style="float: left;"><img src="https://static.igem.org/mediawiki/2015/7/74/WarwickE.coli_Bonded_to_Origami.png" height="300px" width="300px" border="1px"></p>
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The image on the left shows how the E.coli will bond to the DNA Origami structures. We can choose what zinc fingers go on what end of the structures so we could have a pattern in the origami structure. This is useful for analysing microbial communities as it allows different cell types to be brought together.
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An idea we considered was using polymer excreting cells to 3D print structures. 3D bio-printers exist but are used to create living tissue and organs. What we thought of creating was ordinary plastic objects.  The main advantage of this being that the resulting plastic could be bio-degradable, self-sustaining and carbon neutral. Currently there are 8 million tonnes of plastic dumped into oceans each year, which is set to double in the next 10 years and will be tenfold by 2020.
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<br>It would be possible to create 2D and 3D structures using these Origami structures as a glue to hold the cells together but would require hundreds of different zinc fingers to prevent the wrong parts being bonded to one another.
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<br>This shows how a simple shape could be made by using E.coli (black squares) by connecting them with DNA origami (red crosses). In order for a shape to be made each piece of E.coli needs to express a different zinc finger so that it can only be bonded to a specific piece of origami (no non-specific bonding).
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<br>We only have four zinc fingers which means that we don’t have many options for patterns we could make, but given enough time and resources we could easily optimise more zinc fingers so more complex shapes could be made.
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<br>Future iGEM teams could create more zinc fingers which could be combined with our structures so that as time prgresses a database of different shaped and sized oligonucleotide adehsives can be made. Our project could then be used as a stepping stone to create complex 2D and eventually 3D shapes and structures.
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<h5 class="sidebartitle">DNA Origami</h5>
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<p>____________________________________________________________________________________________________________________________________________________</p><h5>Fused Deposition Modelling</h5>
 
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<img src="https://static.igem.org/mediawiki/2015/9/94/WarwickDna_origami_ic.png" class="pics" alt=""> This shows how the DNA strands come together. Three double stranded strings of DNA are denatured and then when slowly cooled will come together to form the Y shape. However after the denaturing each strand of DNA has an equal chance of bonding to the original piece of DNA as it does to the correct origami side. Therefore the more complex the structure the less likely it is that that structure will fully form.
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<p style="float: left;"><img src="https://static.igem.org/mediawiki/2015/4/44/WarwickY_PlasmidSequences.png" height="300px" width="300px" border="1px"></p>This is a sequence we came up with for a Y shaped origami structure. <br><br>
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<br>The highlighted colours correspond to half of one arm which is complementary to half of another arm of the same colour. At the ends of each coloured string is the binding site for a zinc finger.<br>
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<br>This structure will self assemble into a shape with arms of length 150 base pairs with the 9 base pair long binding site on the ends.
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<br>This sequence had to have various boundary conditions, such as reasonable CG content, so that the melting temperature isn't massively out of the required range. The strings also couldn't be allowed to form secondary structures. <br><br> 
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We thought of printing cells into the desired shape using fused deposition modelling (adding layers of cells to create a structure). Once it has been printed the cells would then secrete a plastic which would form the shape. This would have the advantage over normal FDM printers which produce anisotropic materials with an underlying weakness in the z-axis, between the layers of plastic which is the biggest limitation of the technology. This weakness is caused by a lack of adhesion between already partially solidified plastic layers, this stops structurally strong items being made. Our proposed method would eliminate the directionality of strength and would create a structure of uniform strength and flexibility.<br>
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However the size of an <i>E.coli</i> cell is larger than the diameter of the smallest plastic filament possible, so the resulting structure may be less detailed, however if we developed a cell with a small enough radius this problem will be subverted.
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<h5 class="sidebartitle">Minimum Size of Plasmids</h5>
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<br>The image to the left shows how FDM works in regards to plastic.
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It is paramount that the length of the plasmid arms are kept to a minimum length as the longer the arms the more unstable the resulting structure will be. It would also take a longer time to form and would have a lower probability of formation. However if the plasmid arms are kept to the smallest possible size it decreases the likelihood of the correct number of E.coli cells bonding to the ends (we have assumed that the ends of the E.coli are perfect spheres and will bond in the centre- if this is not the case the you will need an extra length to accommodate. We calculated 30% would be the optimum error margin to add). <br> Obviously calculating the plasmid sizes is very important then as it dictates cost and efficiency. The cube construction page explains how this was done.
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<h5 class="sidebartitle">Probability of Formation</h5>
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As you can see the probability of a structure fully forming decreases exponentially as the complexity increases. However, even though for larger number of arms there is a very high chance of a structure forming it is unlikely for all the arms to form. Therefore, for our experiments it would be better to focus on using structures with fewer number of arms to save time and money.
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<h5 class="sidebartitle">Origami Alternative</h5>
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<p>____________________________________________________________________________________________________________________________________________________</p><h5>Stereolithography</h5>
 
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The previous design, which used DNA Origami required lengths of DNA to be synthesised. This is very expensive and time consuming. In order to minimise costs we need to be able to make the structures using already available DNA.
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Another method of 3D printing is stereo-lithography, which works by creating a vat of plastic, in this case cells which then has two lasers, above and below which move and cause the plastic to harden (or cells to react and secrete plastic). The benefit of this is that you can create far more complex and detailed shapes.  Shapes could possibly be made with precision at a cellular level, this sort of accuracy would be of paramount importance for the creation of heart valves and even a bone structure with the correct fibre arrangement. <br>
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<br>As you can see from the images above to accomplish this we will cut correct lengths of DNA out of already available plasmid inserts then denature them, binding two single stranded pieces of DNA together to form a string of twice the length. Doing this allows origami to be used to form a structure as the sides of the single strands will have a complementary pair. This also allows for quick PCR.
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However it would require larger amount of <i>E.coli</i> cells to be held together without dying, a very large challenge to overcome. For this we would use <i>E.coli</i> which would secrete plastic once it is subjected to a certain wavelength of light which would be provided by the two lasers. <br>
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<br>We decided to use plasmids from the last two years iGEM part registry.
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We also though of using a process similar to selective laser sintering, where a film of <i>E.coli</i> cells are spread onto a surface then a laser ‘draws’ the first layer. A second layer of cells is spread on top and the process is repeated. This process takes longer but could be used to make much larger structures.
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Latest revision as of 20:42, 17 September 2015

Warwick iGEM 2015

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