Team:Richmond UR/Results
Results
Above, this gel shows when we successfully made PCR products of pn25
Above is an example of when a gel fails because we barely made any product.
BioBrick part BBa_J04450 cut with S&P
uncut agrobacterium
Ligation plates for pn25, attempt 1
Streaks from Ligation of PN25 plate, attempt 1, fail because the red should not be present
Explanation of Plates/Gels:
Our intention was to create a system in agrobacterium wherein the PN25 promoter would constitutively produce Curdlan. Agrobacterium already produces Curdlan naturally; thus, the genes for Curdlan are already present in the C58 variant of agrobacterium that we utilized in our experiments. However, the promoter of the Curdlan genes is not ideal for our goal of constitutive expression. We needed to create a shuttle vector that housed PN25 and would then be “swapped” for the Curdlan promotor (Pcurdlan) via homologous recombination.
Our primary aims and steps:
1.BioBrick part BBa_J04450 cut with EcoRI & PstI Restriction Enzymes
As a check, the mCherry region should be cut out when BBa_J04450 is cut with EcoRI & PstI and future colonies should not be red
2.Create several oligonucleotide segments that will form the insert
3.Ordered from Invitrogen in 6 segments and then re-combined post-shipping
4.Ligate the vector and insert to create new BioBrick system with PN25 (our target promoter that we want to be in agrobacterium in place of the Curdlan promoter)
5.Ensure the BioBrick PN25 was successfully created via plating on antibiotic plates
6.In our case, at this point, our colonies were all red, indicating an error in the creation of the BioBrick PN25
7.Over the course of the next few weeks, several attempts were made to correct the problem, but it persisted
Finally, using a shuttle vector, inset the PN25 promoter into the genome of a disarmed or non-virulent form of Agrobacterium
As a backup, we ordered Shuttle Vector parts from Twist Bioscience, which are still in testing