Difference between revisions of "Part:BBa K1582025"

Latest revision as of 02:37, 25 September 2015

Thc_Cut1+sJanus-m Fusion Protein

Sequence and Features

Usage

This fusion protein is designed to degrade PET into terephthalic acid and ethylene glycol more powerfully. Compared to single Thc_Cut1, the rate of PET emzymolysis is supposed to be improved by Thc_Cut1-Janus fusion.

Biology

Thc_Cut1 is a kind of cutinase, the details of which can be seen at BBa_K1582028. Class II hydrophobins sJanus are small secreted fungal proteins which can be found in filamentous fungi, which play a role in a broad range of processes in the growth and development of filamentous fungi. Their assembly shows thread-like structure. They could be expressed in prokaryotic cells like E.coli. We did some mutations to it, and we call it sJanus-m. According to some research, when Janus is added to the reaction of cutinase and plastics, the reaction rate is supposed to have an obvious improvement.
Based on the above background, we design to use the method of fusion protein to combine cutinase and Janus compactly, through the establishment of fusion protein, we can found if we can further promote the stimulation rate of the plastics degradation. Meanwhile, from the data about fusion protein, we can try to uncover the functional mechanism of the stimulation to the plastics degradation which is unknown until now.

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-sJanus-m pre-expression. Labels are the same as before.

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-sJanus-m 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

Further exploration

Overview

We successfully build a fusion protein which attach cutinase Thc_Cut1 to our Janus (sJanus-m). The fusion protein has much more better performance than the cutinase before.

Result

The procedure in this part is likely to the pre-experiment, we use different concentration of fusion protein. And we compare the data to the value we conduct with Thc_Cut1 before, found that fusion protein hydrolysis more, especially in high concentration.

Figure 3. These curves compare fusion protein’s performance with normal cutinase in different concentration. We can see that fusion protein perform much better when concentration are over 1mg/ml.

We also conduct our experiments in different pH by using different Tris-HCl. Just as picture shows, the activity of our fusion protein doesn’t change a lot in different pH which is similar with Thc_Cut1.

Figure 4. These two proteins are stable when pH changes, especially when pH is between 6.0 and 9.0. And the concentration of Thc_Cut1 and Thc_Cut1-sJanus-m are 0.2mg/ml.

We drew a curve about detect time and compared it with Thc_Cut1, we can see the hydrolysis rate rose sharply after 5h,while Thc_Cut1 still in the low level. Which represent our Janus works well!
And our fusion protein does improve a lot!

Figure 5. We detect absorbance of liquid every 3h, red line refers to our fusion protein, and the blue line is former enzyme. The performance of cutinase are highly improved when fuse to the sJanus-m.

Figure 6. The picture shows 5mg/ml enzyme have react with one piece of plastic（3*3*0.25mm）for 3h, before and after centrifugation. We can see clearly in the pic. After 3hs the tube of Thc_Cut1-sJanus-m had much more sediment than Thc_Cut1.