Team:KEYSTONE/Basic
We've submitted two basic parts:
1. Part:BBa_K3478888 , LCC-ICCG
2. Part:BBa_K3478890, bLIS
We've characterized and added information to one basic part:
1. Part:BBa_K2753002 , GPPS
1. LCC-ICCG (Leaf-branch compost cutinase) Part:BBa_K3478888
Part type
Coding
Description
The leaf-branch compost cutinase (LCC) is an enzyme capable of Polyethylene terephthalate (PET) degradation that outperforms PET hydrolase and BTA while having a high thermostability. Further site mutation allowed for further improvement in the activity and thermostability of LCC. The LCC-ICCG used in our experiment, which refered to F243I/D238C/S283C/Y127G, is created by Tournier et al. in 2020.This mutation was among the 25 out of 209 that has 75% or more increased activity compared with the original wild-type LCC. The ICCG mutatation was finally developed as a result of a combination of mutations based on the original variant I among the 25. The thermal stability of LCC-ICCG is maintained without the divalent-metal-binding site with the presence of the disulfide bridge. According to experiments, the LCC-ICCG is the most effective at degrading plastic in bioreactors under pH8 and 72°C condition. Furthermore, the leaf-branch compost cutinase is a very effective enzyme as the terephthalic acid (TPA) and ethylene glycol (EG) formed as a result of PET degradation can be used to make new PET plastic. The resulting plastic has similar properties to the PET plastic from factories [1].
Figure 1: (a) The LCC-ICCG gene was coded onto the expression vector pET28a(+) with a 6xHis-tag on both side of N-terminal and C-terminal for more effective purification on Ni-NTA column. (b) The plasmid was transformed into E.coli BL21 (DE3) for LCC production and PET degradation to produce terephthalic acid (TPA) and ethylene glycol (EG) which can be used to produce new PET.
LCC Production & Purification
Figure 2: Characterization of LCC-ICCG. (a) DNA gel electrophoresis of pET28a(+) plasmid coded with LCC-ICCG and transformed in E.coli BL21. (b) Protein gel electrophoresis of LCC expression with 300µM IPTG and purification with 20mM Tris-HCL, 300mM NaCl buffer, showing a molecular mass of 28kDa as expected.
Out of all conditions tested, we were able to produce an optimal yield of 27.3mg/L of LCC with 300µM IPTG induction and purification with 20mM Tris-HCL, 300mM NaCl buffer. By increasing the concentration of NaCl in the buffer from 100mM to 300mM, we were able to increase the yield from 4.22mg/L to 27.3mg/L, as this concentration of NaCl better stabilizes the structure of LCC [1]. However, this yield has been affected due to the considerable amount of LCC proteins that has been remaining in the cell lysis precipitant as shown in figure 2.b, which could have been caused by the following reasons: a) the ultrasonic lysis wasn’t entirely complete/effective, and some bacteria remained intact; b) inclusion bodies formation.
PET Degradation [HPLC]
Since the ICCG variant of the LCC enzyme has an increased thermostability, it is able to achieve optimal degradation at a temperature of 72˚C [1], which allows PET to reach glass transition state and become amorphous, enabling more effective degradation [2] (see more from prof. Liu’s interview section.) We repeated the degradation experiment by Tournier et al. with the LCC proteins that we produced, using 1 ml of a 0.69µM solution of purified protein in 20 mM Tris-HCl, pH 8, 300 mM NaCl, and combined with 100 mg PET powder with 49ml of 100 mM potassium phosphate buffer pH 8 in a 100 ml flask for degradation in a 72˚C environment under 170 rpm agitation. In addition, we tested the effectiveness of degradation with an extended range of LCC concentrations in the 1ml solution, from 0.25µM, 0.5µM, 0.69µM, 5µM, 10µM, 15µM to 20µM, as well as a controlled sample of 100mg PET powder in 1ml of 20 mM Tris-HCl, pH 8, 300 mM NaCl and 49ml of potassium phosphate buffer pH 8.
Figure 3: Gas chromatography test for ethylene glycol in LCC degradation supernatant with different LCC concentrations after 24h. *Note: the concentration refers to the concentration of LCC in the 1ml solution that is added to 49ml buffer.
After 24h of degradation, we sent the supernatant samples to gas chromatography testing to test the concentration of ethylene glycol, a product of PET degradation, to examine the effectiveness of LCC in degrading PET. Although a wide range of samples with different concentrations were tested as stated above, a number of them have been mysteriously lost in the process of transportation for GC testing, therefore we were only able to produce the results as shown above. The standard sample used in the GC test was 20mg/ml EG. In our sample of 100mg PET in 50ml buffer, the maximum concentration of EG that can be obtained is 0.54mg/ml, which would produce a peak with an area of 0.27% of the area of the standard peak. Therefore, although difficult to obtain a precise calculation, the amount of PET degraded by 5µM and 10µM of LCC was significant, and a distinct difference with the no LCC controlled sample can be seen. The results might have been affected by the fact that the temperature used for degradation experiment was 70˚C instead of 72˚C due to equipment limitations.
Source
Site mutation from wild-type LCC.
Design considerations
We chose to use the ICCG mutation (F243I/D238C/S283C/Y127G) out of all mutations of wild-type LCC because it displayed the best overall performance, taking into consideration of their thermostability and enzyme activity, according to Tournier [1].
2. bLIS (bLIS(3R)-linalool synthase) Part:BBa_K3478890
Part type
Coding
Summary
bLIS is a gene that expresses linalool synthase The linalool synthase used in our experiments is the linalool synthase from Streptromyces clavuligerus. linalool synthase is a enzyme that can effectively synthesize linalool
Description
bLIS is a gene that expresses linalool synthase The linalool synthase used in our experiments is the linalool synthase from Streptromyces clavuligerus. linalool synthase is an enzyme that can effectively synthesize linalool. we used Gibson assembly to combine bLIS fragment and its vector pR6K, and transformed it in to E. Coli DH5α, and linalool synthase has successfully been produce.
we used pTac promoter and lacI repressor in the plasmid, which means that IPTG can be used to induce the production of linalool synthase.
Figure 1: protein electrophoresis results of linalool synthase (65.6 kDa)
However, coding bLIS need to work cooperate with GPPS and MVA which produces the substrate, GPP, for bLIS. We used Gibson assembly to produce plasmid pR6k-ptac-GPPS-bLIS and p15A and MVA. we transformed both plasmids into E.coli DH5α and conducted a colony polymerase chain reaction of E.coli transformed with pR6K-ptac-GPPS-bLIS, which allows us to determine the success of Gibson assembly (figure 7A). Therefore, we successfully constructed and transformed pR6K-ptac-GPPS-bLIS to E.coli DH5α.
Figure 7: results of Gibson assembly. (A) gel electrophoresis result of pR6K-ptac-GPPS-bLIS (14358 bp). (B) gene sequencing result of p15A-MVA.
We cotransformed P15A-MVA and pR6K-ptac-GPPS-bLIS plasmid to E.coli DH5α. E.coliDH5αwe induced them with IPTG and two of them with extra glucose. after the inducing process, the bacteria solution was treated with n-hexane to extract purified linalool(figure 9). We can clearly smell the strong fragrance of linalool in those samples, and samples with glucose have a stronger fragrance than samples that are not treated with glucose. This indicates that glucose may promote the synthesis of linalool in E.coli DH5α. We do not need to knock out the gene in E.coli producing stinky smell because the aroma overcomes the smell of E.coli. Therefore, our E.coli won’t affect the local environment and appearance.
Besides, we also compared the smell of our sample to standard linalool sample, and standard geraniol, which is the product of pR6K-ptac-GPPS-GES (the plasmid before our improvement) Based on our observation the aroma of geraniol contains sweetness; whereas, the aroma of standard linalool sample has a slight peppery smell. The two smells are quite distinguishable. The small of our samples also have a slightly peppery aroma, which is close to the linalool standard sample. Thus, we have successfully produced linalool.
In addition, through visual observation, we also confirmed that glucose can promote the synthesis of linalool in E.coli. The transparent liquids in the test tubes are purified linalool (figure 9). The volume of the liquid in samples treated with glucose is larger than samples that aren’t treated by glucose. Therefore, E.coli treated with glucose may be able to produce more linalool in the same condition than E.coli that is not. Our next step may be finding the optimal condition for linalool synthesis in E.coli. However, due to the Covid-19 situation, we do not have enough time in the lab, so we can only do those experiments in the future.
Figure 9: E.coli DH5α with P15A-MVA and pR6K-ptac-GPPS-bLIS was propagated in four separated conical flasks. After the bacteria solutions reach the suitable OD (0.6-0.8), we added 2.5 ml glucose to two of the mediums during and added 25mM of IPTG to By adding n-hexane (4.5ml) to the induced bacteria solution, we can extract pure linalool (transparent layer)
References
[1] Tournier, V., Topham, C.M., Gilles, A. et al (2020). An engineered PET depolymerase to break down and recycle plastic bottles. Nature 580, pp.216–219. https://doi.org/10.1038/s41586-020-2149-4.
[2] Luo, L. (2020). Personal Interview.
