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Team:Imperial College London/Project/Switch/Results
From 2011.igem.org
Chapter 1: Assembly strategy
The assembly of this module shall be the most challenging out of all of them. Not only are we starting it the latest, but we will be using parts from the registry to assemble it. The first step of assembly will require us to place the anti-Holin from the BBa_K112808 biobrick under the J23100 promoter in BBa_K398500. In order to perform this step we will be using a PCR which will contain non-homologous regions. These non-homologous sequences will contain the insulator, RBS (ITR obtained from modelling) and 15bp overhangs that will allow us to assemble the PCR products of both the biobricks through the use of In-Fusion. The PCR step will be incredibly challenging. Once the parts are correctly inserted into the pSB1C3 vector we will be able to extract it and use biobrick assembly to insert it into the Crim plasmid. Once in the Crim plasmid, the gene must be integrated into the genome. Once this step is completed we can proceed to the transformation of these cells (any attempts at transformation before we have these cells will just result in cell lysis).
We will also require the use of the J23103 promoter (the RPU which we have obtained from modelling)which can be found in a BBa_J61002 vector. We also have ordered an oligo of the promoter to run in parallel. Once this has been inserted into a pSB1C3 plasmids, we can extract the Holin and Endolysin genes from the BBa_K112808 biobrick using primers that will contain a SpeI or a PstI site for biobrick assembly. Once the J23103 is assembled with the Endolysin and Holin we can transform the E. coli that contain the anti-Holin gene in the genome.
Time is running out. Will this module be completed? We bloody hope it will.
3rd of August
Today we attempted to transform 5α cells with the BBa_K112808 kill switch cassette. These cells will be incredibly important for later steps in the assembly process.
4th of August
Today we performed a successful mini-prep on the previously transformed cells. This DNA is now ready for subsequent assembly.
8th of August
Today we attempted a transformation of cells with the BBa_K093005 biobrick. We will be using the RFP in this plasmid in order to make sure that the final constructs contain both the integrated Crim plasmid (contains GFP) and the pSB1C3 with the Endolysin (will contain the RFP).
9th of August
Today we performed a successful mini-prep on the previously transformed cells. This DNA is now ready for subsequent assembly. However, in order to proceed we will need to know the expression ratio between the genome promoter and the plasmid promoter. We have to make sure that the amount of anti-Holin is only slightly higher than the level of Holin as to not exhaust the cells too much. This is pretty difficult considering that one of the genes will be in a high copy plasmid whereas the other will be in the genome.
16th of August
Today we transformed the cells with the BBa_J61002 vector containg the J23103 promoter that will be needed for the plasmid.
18th of August
Today we attempted to run the digested gels. Unfortunately, the gel that was used had lost its Sybr safe somehow overnight meaning that no bands could be extracted. However, we were told that we have access to an oligo of the J23113 promoter which has a similar strength to the J23103 promoter. We shall attempt to use this promoter as well before the ordered oligo of J23103 arrives.
19th of August
Today we prepared the vector for the promoter insertion by digesting it with EcoRI and XbaI and then gel purifying the sample. We should be able to ligate the J23113 promoter into BBa_K093005 on Monday (provided that the gel extraction worked...)
