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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...)
22nd of August
The primers that we need for the assembly has arrived today. We prepared a PCR using the "short" primers. These allowed us to make the templates that we need for the "long" primers (contain large non-homologous regions that will allow us to use In-Fusion later on). We then PCR'd the Anti-holin with the template. Hopefully the PCR worked and we will be able to do a cheeky MlyI digest tomorrow.
23rd of August
The gel we ran today on the Anti-holin PCR product has given us an excellent yield of DNA which we will be able to use for the In-fusion attempt.
From left to right: Ladder from Baldwin, Anti-holin PCR, Anti-holin PCR, Ladder from Invitrogen.
We also performed the short and then the long PCR on the pSB1C# vector containing the J23100 promoter.
25th of August
We have been having problems obtaining a decent yield for the long J23100 PCR. We attempted to run another PCR again overnight in order to recitfy this issue. Today we also determined that the CRIM plasmid is missing either an XbaI site or a SpeI site. Depending on which one it is missing we will have to integrate the Anti-holin upstream or downstream of the uGFP within the plasmid.
From left to right: EcoRI+PstI; XbaI SpeI.
26th of August
We finally managed to obtain a yield of DNA larger than 10ng/μL with which we can attempt the In-Fusion reaction as well as the CPEC reaction. However, the yield might still be too low meaning that we will have to repeat the PCR once again using a different number of cycles and temperatures. We also ran another digest to determine which recognition site is missing on the CRIM plasmid. From the gel we can assume that the missing site is SpeI.
From left to right: GFP digested with EcoRI+PstI; XbaI+SpeI; Ladder; CRIM digested with XbaI; SpeI; XbaI+SpeI
6th of September
Temperature gradient PCR of superfolded GFP to get back Spe site. Reactions 1 and 4 worked and will be Dpn1 digested and PCR purified and then the concentration will be measured.
Chapter 2: Modelling to guide the Bactrap design
Modelling of BacTrap is important for us to choose appropriate promoters and ribosome binding site (RBS) to inhibit and optimize lysis behavior in our genetic modified cell and soil bacteria respectively. Therefore we created a steady-state kinetic model to describe this.
In wild type soil bacteria, cell lysis will be induced when the genetic modified plasmid transmit to them. We assumed there would be 50 copies of plasmids get transferred to one soil bacterium.
From literature, the cell lysis happens at holin level of 1000 per cell. Therefore, the critical concentration of holin was set to be 1000/cell. The ODEs of holin expression in one soil cell is illustrated below:
At Steady state:
In the genetic modified bacteria, the promoter strength and RBS strength of anti-bolin should be stronger than holin such that all the holin produced by 300 copies of plasmids will be inhibited by antiholin.
The anti-holin can bind to holin to form anti-holin holing dimer (AH-H), which is defined as deactivated holing. The equations describe this mechanism are listed below:
At Steady state:
where PH represent the holin mRNA promoter strength and PAH represents the antiholin mRNA promoter strength. KH and KAH are the RBS strength of holin and anti-holin. γmRNA,H and γmRNA,AH represent the degradation rates for holin and antiholin mRNAs respectively and γ represents the protein degradation rate. kH and kAH represent the rate constants for holin and antiholin protein translation and kc and ku represent the coupling and uncoupling rates for the holin-antiholin dimer.
By choosing value of m such that the concentration of holin is kept at a very low level (<< 1000/cell) can assist in the optimization of kill switch design. Plot the steady state concentration of holin against different values of m, it is shown that there will be no holin production as long as m greater than 300.
choose m = 400 and tested that m value in the ODEs described above. All the holin in GM cell are inhibited and holin concentration in wild bacteria will reach 1000 in short future.
