Difference between revisions of "Team:Warwick/Modelling1"

Revision as of 10:31, 19 August 2015

Warwick iGEM 2015

DNA Origami Glue

DNA Origami

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.

Minimum Size of Plasmids

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. Below explains how this was done.

Probability of Formation

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.

Origami Alternative

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.

Design by Warwick iGEM 2015