This deals with the issue of creating 2D and possibly 3D shapes without excessive waste of DNA. It does this by sticking cells together using DNA Origami as a glue. DNA origami is a method of creating shapes out of DNA by designing strands of DNA to be complementary to one another so that when they are put together and denatured and annealed they form a shape.
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.
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.
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).
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.
Future iGEM teams could create more zinc fingers which could be combined with our structures so that as time progresses a database of different shaped and sized oligonucleotide adhesives can be made. Our project could then be used as a stepping stone to create complex 2D and eventually 3D shapes and structures.
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.
This is a sequence we came up with for a Y shaped origami structure.
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.
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.
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.
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).
Obviously calculating the plasmid sizes is very important then as it dictates cost and efficiency. The cube construction page explains how this was done.
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.
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.
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.
We decided to use plasmids from the last two years iGEM part registry.
DNA Origami Glue Sequences
Below shows the process by which the DNA origami structures are made using already available DNA.
The right shows the sequences and linkers which we will use.
Procedure
1) Remove insert from plasmid
2) PCR the plasmids
3) Use restriction enzymes to cut the plasmids into smaller pieces of equal length (150 bp)
4) Create "linker" DNA
5) Denature the double stranded DNA and add linkers
6) Allow to cool and be amazed at the results
