Team:Imperial College London/Project/Switch/Results

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.

Modelling of Gene Guard

Introduction

The gene guard device involves three proteins:

1) Holin is a protein forms pore complexes in the inner-membrane and this pores allow endolysin to move into periplasmic space.

2) Endolysin is an enzyme that could break down the cell wall and causes cell lysis.

3) Anti-holin binds to holin and prevent from forming pores.

Our system will have holin and endolysin genes together with axuin genes and chemoreceptor genes on the plasmids, and anti-holin genes on the genome under control of strong promoter. The production of anti-holin will prevent cell lysis of the genetic modified bacteria by deactivating holin. If the plasmid get horizontal transferred to a different cell without anti-holin on its genome(e.g. any wild type soil bacteria), the expression of holin and endolysin will induce cell lysis and hence prevent it from keeping the plasmid containing auxin gene.

The key to the success of this device is the right ratio of holin and anti-holin production such that the anti-holin can deactivate all the holin produced in our genetic modified bacteria, and the holin expression level in wild type bacteria is high enough to induce cell lysis. As the gene expression is controlled by both promoter strength and ribosome binding site (RBS) strength, therefore modeling of gene guard is important for helping us to choose appropriate promoters and ribosome binding site (RBS) to inhibit and optimize cell behavior in our genetic modified cell and soil bacteria respectively.

Modelling Design

The modeling of gene guard consists two parts, one is the holin production in wild type bacteria and the other part is the deactivation of holin by anti-holin in genetic modified bacteria. Since all the genes in our system are constitutively expressed, thereby we only consider steady state.

Holin production in wild type soil bacteria

This part of modeling focused on deriving the relationships between promoter strength and RBS strength of holin on single plasmid. In order to model this, the following assumptions were made:

1) After plasmid transfer and duplication, there will be 50 copies of plasmids in one bacterium.

2) The literature indicates that cell lysis happens at holin concentration equals to 1000/cell[1].

Based on above assumptions, the kinetics of cell lysis in wild type bacteria was modeled using ordinary differential equations (ODEs) for the holin mRNA and holin (Equation 1).

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.