Our Team has planned several experiments to confirm each parts we designed, the our design and engineering process is illustrated by the flowchart below:
We have designed the CRISPR Cas9 single guided RNA for knocking out arcA and iclR genes in the E. coli genome. Their sequence and part design can be found in K3718003 & K3718004.
Transform sgRNA and Cas9 into wild type E. coli
sgRNAs for arcA and iclR will be contained in a plasmid with CRISPR Cas9 gene. The plasmid will then be transformed into a wild type E. coli by heat shock transformation.
SDS PAGE/ Diagnostic PCR
After transformation, SDS PAGE or colony PCR will be done to confirm whether the E. coli is successfully knocked out. Forward and reverse PCR primers will be designed for arcA and iclR.
If the E. coli is not knocked out, we may have to redesign the sgRNA or repeat the transformation of the plasmids.
Cell growth assay
After the E. coli is sure to be knocked out, we will wait for the plasmids to be lost after generations of cells. To check if the cell growth is reduced after the genes are knocked out, a cell growth assay will be done to compare the growth rate of WT E. coli and K.O. E. coli. We will design the sgRNA again if the cell growth of K.O. E. coli is not lower than that of WT E. coli.
Transform MRP into wild type E. coli
Plasmids containing MRP gene will be transformed into normal strain E. coli before directly transforming them into the K.O. strains. The sequence of MRP can be found in K3425100.
SDS PAGE will be used to test if the MRP is expressed. The transformation will be redo if the bends are not shown in the SDS PAGE.
Transform MRP into K.O. strain
If the protein expression is successful, the MRP gene will be transformed into the K.O. strain to make sure the MRP protein can also be expressed in the K.O. E. coli.
SDS PAGE will be used to test if the MRP is also expressed in the K.O. E. coli. The transformation will be redo if the bends are not shown in the SDS PAGE.
Transform rescue genes into K.O. strain
While the transformation of MRP is doing, plasmids with iclR and arcA gene will be transformed into arcA-iclR- E. coli as rescue genes.
Cell growth assay
Cell growth assay will be done to compare the growth rate of wild type E. coli, iclR- E. coli, arcA- E. coli, arcA-iclR- E. coli and rescued KO E. coli. If the growth rate of the rescued E.coli is lower than that of KO strains, we may need to redo the transformation step.
Caffeine as model
After the experiments of MRP expression and rescue KO E. coli have proven to be successful, we will use caffeine-sensitive MRP (K3718010) as a testing part to model the system. Caffeine will be used to homodimerize the receptors, and is expected to induce the expression of downstream genes. This model will be used to conduct three experiments to confirm the functionality of the tool. By cloning in an antibody VHH gene against caffeine and gfp as a desired gene to be expressed, we expect the platform is activated in the presence of caffeine to express gfp, arcA and iclR, which will result in an increase in growth rate and a visible green color under UV.
Cell growth assay
We will conduct a cell growth assay to compare the growth rate of wild type E. coli, iclR- E. coli, arcA- E. coli, arcA-iclR- E. coli, rescued E. coli and the E. coli with the MRP (caffeine) and rescue genes.
We will measure the leak expression of MRP’s downstream genes before adding caffeine to let MRP bind to the CadC region.
We will measure how much concentration of caffeine is needed for MRP to detect and express the downstream genes effectively. This is tested by adding different concentrations of caffeine to our final kit and find the optimum concentration to induce MRP.
The growth rate of arcA-iclR- E. coli is what we would be model for, since our lab does not allow us to knock out the E. coli genome by ourselves. We will use the growth parameters of arcA- and iclR- E. coli to predict the growth parameters of arcA-iclR- E. coli.
For the details of modelling, please go to [modeling].