Part:BBa_K1582028
Thc_Cut1: Cutinase 1 from Thermobifida Cellulosilytica
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
Cutinases are well known for their ability to depolymeraze cutin and water-soluble esters like p-nitrophenyl esters and insoluble triglycerides. They could also degrade PET the plastic.
Cutinases Thc_Cut1 is from Thermobifida cellulosilytica. We use it in our project for PET emzymolysis. Meanwhile, we also made Thc_Cut1-Janus fusion protein to make enhance cutinase’s activity.
Protein Expression
Protein pre-expression experimental conditions:
1.Transferred our correct plasmid into escherichia coli BL21 (DE3).
2.Select bacterial colony and add into LB, incubate at 37 centigrade for 7h. Add 4μl of IPTG to induce the expression of protein.
3.Incubate at 37 centigrade for 4h. The bacterial solution’s OD is 0.6-0.8.
Figure 1. The result of protein Thc_Cut1 pre-expression. 1: bacterial No.1 non-incubated; 1’: bacterial No.1 incubated; 2: bacterial No.2 non-incubated; 2’: bacterial No.2 incubated; 3: bacterial No.3 non-incubated; 3’: bacterial No.3 incubated
Finally, the protein was expressed successfully.
We added 5μl of the bacterial restored into the LB containing 5μl of ampicillin to the final concentration of 1mM. And then, we incubated them in the shaker at the temperature of 37 centigrade, working for 14-16 hours.
Experimental conditions as follow：
1. Incubate at 37 centigrade, until OD ranges from 0.6-0.8(4-5h).
2. Incubate at 4 centigrade, 220rpm, for 30mins.
3. Add 1mL IPTG to the final concentration of 1mM.
4. Incubate at 16 centigrade for 12-16h.
The purification of protein:
We used eppendorf to make bacterial deposited, working at 4000rpm for 20min. Use 15mL MCAC0 to suspend bacterial.
After resuspending, we used high pressure to break the cells.
In order to get the recombinant protein with the higher purity, the recombinant protein was purified through Ni-chelating affinity chromatography.
The results are as follow:
Figure 2. The result of protein Thc_Cut1 expression. 1: Sample non-induced; 2: Sample induced; 3: Sample of cytoplasm deposited; 4: Sample which outflowed from Ni column combined with supernatant; 5:Sample of NIi medium combined with supernatant; 6: Sample washed by MCAC20; 7: Sample resuspended by MCAC30; 8: Sample washed by MCAC30; 9: Sample resuspended by MCAC50; 10: Sample washed by MCAC50; 11: Sample resuspended by MCAC100; 12: Sample washed by MCAC100; 13: Sample resuspended by MCAC200; 14: Sample washed by MCAC200; 15: Sample resuspended by MCAC500; 16: Sample washed by MCAC500; 17: Sample resuspended by MCAC1000.
Stimulated Plastic Ezymolysis
Pre-experiment
Overview
We have found out appropriate concentration of the cutinase we made and suitable pH for our enzyme Thc-Cut1, the right time to detect the product of hydrolysis of PET.
Result
We use absorbance in 600 nm to measure the turbidity of the liquid in tube. We tried different concentration of enzyme with one piece of plastic, decided to use 2mg/ml to conduct our experiment afterwards.
Figure 8. This curve describe the OD600nm after 3h’s reaction changes through the concentration of Thc_Cut1. We can see clearly at 2mg/ml, the curve reaches a peak, at which concentration we will compare the hydrolysis effect.
We also conducted our system in various pH, found that the activity of Thc_Cut1 doesn’t change much in different pH, so we chose pH 7.0 as one of our experimental condition.
Figure 9. The curve shows the absorbance at 600nm changes along with pH. The activity of enzyme doesn’t change much in various pH.
In order to find right time to detect, we detect OD600nm in several time, here’s the time line.
Figure 10. The timeline clearly indicates the hydrolysis rate rises along with time.
Considering we need an appropriate detect time to measure the hydrolysis effect, we set our detect time at 3h.
Main experiment
Overview
We discovered the right temperature to conduct various way to mix up our sJanus (or inJanus) and Thc_Cut1.And we have found out the best way to increase the hydrolysis rate.
Result
We conduct our trail in different temperature ,realizing 50℃ is the proper temp for the enzyme and our Janus to combine.
Figure 11 Different color represent different ways of mix. Thc_Cut1+Janus means enzyme had incubated with Janus for 16h before the plastic was added into the system. And Plastic+Janus means plastic had incubated with Janus before adding enzyme. And we also conduct these incubation separately in 4℃,37℃.50℃, values at 50 degree are higher than that under the other temperature.
Based on the experiment above, we compared these two different ways with simple mix, here’s the result.
Figure 12. The tag with “*” means simple mixture. We can conclude that pre-incubation does increase the hydrolysis rate, some ways(Plastic+sJanus) even boost 200%+ react rate comparing with Thc_cutL1* in our conditions!! Another interesting phenomenon is sJanus usually works better than inJanus, and the reason behind it need our further exploration. On the whole, Our Janus works a lot!
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