Due to the global pandemic in 2020, all wet labs of our team have been suspended. The team chose to put our effort on research papers related to our project and draft the following experimental design.
All research work in Phase one aims at checking the expression of the right cutinases by the transformed bacteria.
We would carry out the protein electrophoresis to distinguish the gene products (cutinases) by comparing their relative molecular sizes. The concentration of different cutinases would also be estimated in order to compare at later stages.
The second experiment is to compare the optimum conditions and relative efficiencies of different cutinases obtained. First, we shall check the protein concentration of all the cutinases. This ensures fair comparison of the degradation rate of different cutinases on PET per unit concentration. BCA assay will be used to measure the protein concentration.
A thorough review of all research papers related to cutinases provide us valuable experience on the measurement of degradation efficiency and the material used for degradation. We plan to carry out a series of experiments measuring the degradation efficiency under different pHs, temperature and salinity with the same set-up and measurement methods.
Two measurements will be carried out to measure the result rate of degradation in the second experiment. Method one is to measure the change in weight of the PET film over a fixed period of time. By doing so, we can compare the efficiency of different cutinases and which strain is most efficient. The rate of degradation can be estimated using the following formula:
Formula (% per hour): weight loss % / time(hours)
Here we assumed that the rate of degradation is constant over the course of the experiment.
Method two is to use pH-stat to measure the NaOH consumption. As one of the degradation products, terephthalic acid, is acidic. The NaOH consumed in neutralization indicates the amount of acidic product and hence the cutinase efficiency. Graphs of NaOH consumption versus time of various cutinases will be plotted to reveal their relative efficiencies. It is assumed that the activity of cutinases remains constant despite the change in pH.
To clone the selected cutinase gene, we designed a plasmid consists of 8 parts: the target cutinase gene, his-tag, promoter, terminator, lac repressor, lac operator, multiple cloning site (MCS) and an antibiotic resistance gene (KanR) as selection marker.
The first comes is the cutinase gene that codes for the specific amino acid sequence to make up the enzyme, which is then followed by a 6x his-tag. The his-tag serves as an affinity tag for purification via nickel column chromatography. The expressed his-tagged protein can be purified and detected easily. The cutinase gene and the 6x his-tag are chained together as a single gene of interest and it would be ordered from TWIST Bioscience, with flanking restriction sites SalI and SacI on the ends of the sequence. The flanking restriction sites provide staggered cuts complementary to those in the MCS, hence they are readily joined by DNA ligases.
In front of the gene of interest locates the lac operator, which modulates the expression of the cutinase gene in the bacterial culture and to ensure better yields.
A lac repressor is inserted on the other side of the plasmid so that the lac operator is inhibited when expression is not induced. To induce expression, Isopropyl β-D-1-thiogalactopyranoside (IPTG) will be added. IPTG relieves the effect of inhibition by the lac repressor, thus leading to the expression of the cutinase gene.
Before the lac operator-cutinase-his-tag complex, there is a T7 promoter. T7 promoter is a constitutive promoter, which is active all the time. This means the expression of the cutinase gene is only modulated by the lac repressor. It also serves as an origin of transcription for RNA polymerase. To stop the transcription process, a T7 terminator located behind the gene complex is needed. The terminator stops the transcription so that only our gene of interest is transcribed.
Our plasmid backbone also includes an antibiotic resistance gene (KanR) as a selection marker, its expression makes the transformed bacteria resistant to the antibiotic kanamycin. When the transformed mixture is cultured in a medium containing kanamycin, all untransformed bacteria will be killed and only the transformed bacteria survive and reproduce to form colonies in the culture medium.