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Team:Imperial College London/Project Gene Assembly
From 2011.igem.org
Module 3: Gene Guard
Containment is a serious issue concerning the release of genetically modified organisms (GMOs) into the environment. To prevent horizontal gene transfer of the genes we are expressing in our chassis, we have developed a system based on the genes encoding holin, anti-holin and endolysin. We are engineering anti-holin into the genome of our chassis, where it acts as an anti-toxin, and holin and endolysin on plasmid DNA. In the event of horizontal gene transfer with a soil bacterium, holin and endolysin will be transferred without anti-holin, rendering the recipient cell non-viable and effectively containing the Auxin Xpress and Phyto-Route genes in our chassis.
Assembly
The assembly of this module shall be the most challenging. Not only are we starting it the latest, but we will be using standard Biobrick assembly. The assembly of this module will go through three stages which must be completed sequentially. First we must create the Anti-Holin construct that will contain the insulator and our strong RBS. Then we must insert the Anti-Holin insert into the CRIM plasmid and integrate the plasmid into the genome. Once the Anti-Holin is in the genome we will build the Holin-Endolysin construct that will only be able to be maintained within our strain of E. coli.
Stage 1: Anti-Holin construct assembly
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 PCR to add our new RBS and insulator sequence into the construct. The primers were designed to have 15bp overhangs for In-Fusion. The PCR step was incredibly challenging due to the long non-homologous regions within the primers. This cost us almost two weeks. However, after a lot of hardship and numerous In-Fusion and CPEC attempts we managed to assemble this construct.
Gel image 1: This is a EcoRI + PstI digest of two of the colonies on the Anti-Holin In-Fusion plate that showed promise in the colony PCR's. Lane 1 is the ladder, Lane 2 is Colony 2, Lane 3 is the ladder and Lane 4 is colony 9.
As we can see, colony 2 does not contain the insert we are looking for. However, colony 9 has an insert of the correct size. This stage has been completed successfully.
Stage 2: Integration of Anti-Holin into Escherichia coli genome
The next stage of the assembly is to integrate the insert from our Anti-Holin construct into the genome of E. coli. In order to do that we will ligate the insert into the CRIM plasmid. Once inserted we must transform the plasmid into pir+ cells in order to replicate the plasmid. Once we have a good yield of the plasmid we will co-transform the CRIM plasmid into cells containing the accessory plasmid and we will select for the colonies that contain the CRIM plasmid in the genome.
During this process we discovered that the SpeI site is non-functional due to the non-RFC10 insertion of a terminator. This was also discovered within our sfGFP construct which we had to repair through the use of PCR, phosphorylation and ligation of the plasmid. This has altered our plans to run two insertions in parallel (one in the suffix and one in the prefix) to see if there would be any difference in Anti-Holin expression levels.
Stage 3: Construction of the Holin-Endolysin construct
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 really 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
1st of September
After two unsuccessful In-Fusion attempts we decided to go back to take a look at our primers and found that there was an error in the vector primers. We decided to rectify this by performing a PCR with a short and long primer. This was successful and it gave us a good yield of DNA to work with for the In-Fusion reaction.
5th of September
Today we ran a gel of a CPEC and In-Fusion reaction in order to try to get the Anti-Holin into the pSB1C3 plasmid. The gel showed no results, we will have to continue trouble-shooting and repeating these reactions.
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.
7th of September
Today it seems like the In-Fusion finally worked. There were no colonies on the control plate and there were at least fifty colonies on the In-Fusion plate. In order to prepare for the Anti-Holin in the genome we have started the PCR on the endolysin and holin genes from the kill switch cassette.
From left to right: Ladder; CRIM digest with E+X; Holin+Endolysin PCR product.
8th of September
Today we performed the PCR on the template of the Holin and endolysin we made the other day. This allowed us to add and XbaI site as well as the suffix to the template without much issue. We also digested the CRIM plasmid with EcoRI and XbaI in preparation for the insertion of the Anti-Holin into the genome.
From left to right: Lanes 1-5 are Holin+Endolysin PCR products; Lane 6 is the CRIM plasmid digested with EcoRI and XbaI.
9th of September
Today we performed a colony PCR on the Anti-Holin In-Fusion plate. Most of the results gave us no interest. However, since there was no cells on the control plate we believe it is due to the cells not bursting properly in the PCR reaction. On the other hand, colony 9 gave us a nice band at around the right size.
Top gel: Ladder; 14, 15, 16, 17, 18, 19, 20, Control, Ladder
Bottom gel: Ladder, 1,2,3,4,5,6,7,8,9,10,11,12,13,Ladder
10th of September
We have performed the colony PCR once again. This time colony two gave us a nice clear band of the appropriate size. Colonies two and nine have been chosen for further testing.
