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           <div class="div-center">(a)<img class="big-img" id="F23S" src="https://2021.igem.org/wiki/images/9/97/T--BJEA_China--Resultbu.png"></div>
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           <div class="div-center">(b)<img class="big-img" id="F23S" src="https://2021.igem.org/wiki/images/7/7d/T--BJEA_China--molecularcloning4.jpg"><br></div>
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           <p class="text-ctr">(Fig1. (a). M: marker; Lane 1: mLCC 780bp; Lane 2: mLCC-linker-mHGFI 1059bp Lane 3: mLCC-linker-mHFBI 1035bp; Lane 4: mLCC-linker-BslA 1227bp<br>
 
           <p class="text-ctr">(Fig1. (a). M: marker; Lane 1: mLCC 780bp; Lane 2: mLCC-linker-mHGFI 1059bp Lane 3: mLCC-linker-mHFBI 1035bp; Lane 4: mLCC-linker-BslA 1227bp<br>
 
(b). M: marker; Lane 1,2,3,4: mLCC 780bp; Lane 5,6,7: mLCC-linker-mHGFI 1059bp Lane 8,9.10: mLCC-linker-mHFBI 1035bp; Lane 11,12,13: mLCC-linker-BslA 1227bp
 
(b). M: marker; Lane 1,2,3,4: mLCC 780bp; Lane 5,6,7: mLCC-linker-mHGFI 1059bp Lane 8,9.10: mLCC-linker-mHFBI 1035bp; Lane 11,12,13: mLCC-linker-BslA 1227bp

Revision as of 04:12, 20 October 2021

mLCC


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 193
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage

Our group decide to enhance the activity of mLCC by proceeding two approaches, which are constructing a fusion protein of mLCC and hydrophobins and using the technique of Bacillus subtilis surface display.

The first approach is constructing the fusion protein which was made to enhance the efficiency of adsorption, since the surface of PET film is hydrophobic and the surface of mLCC is hydrophilic. By constructing the mLCC-linker-mHFBI, mLCC-linker-mHGFI and mLCC-linker-BsLA fusion protein, the PET degradation efficiency will be enhanced due to the unique properties of amphiphilicity and self-assembly of hydrophobins.

The second approach is using the technique of Bacillus subtilis surface display. By combining mLCC with the coat protein to form CotB-linker-mLCC, CotC-linker-mLCC, CotG-linker-mLCC and CotC-linker-mLCC fusion protein. mLCC will be immobilized on the Bacillus subtilis cell surface to obtain a recyclable whole-cell biocatalyst, which can reduce costs and make the mLCC more efficient degrading PET.

Biology

LCC is a leaf-branch compost cutinase[1] and a kinetically robust protein[2]. A research published on Nature came up with a mutant enzyme, mLCC[1] that hydrolyzes 90% of PET in plastic bottles in just 10 hours. This is more efficient than any previous PET hydrolase, and more importantly, the resulting monomers- ethylene glycol and terephthalic acid have the same properties as the monomers found in petrochemical materials.

Design Consideration

Create a fusion protein by jointing mLCC with hydrophobins：

The construct was cloned into a pET28a plasmid and transformed into BL21 (DE3) E. coli.

The construction includes:

1. a 6× His tag is added to enable us carrying out Ni-NTA protein purification

2. The CT fused with BslA, mHGFI or mHFBI with a GS linker (three Glycine Serine repeat: GGGGSGGGGS)

Construct Cell surface display of mLCC in Bacillus subtilis：

The construct was cloned into a pHT43 plasmid and transformed into B.subtilis BS168 The construction includes:

1. CotB is fused with mLCC at the NT with a GS linker (three Glycine Serine repeat: GGGGSGGGGS)

2. A flag-tag is added at the C-terminal to provide conditions for the use of fluorescence to detect the target protein after it is displayed on the cell surface.

Joint mLCC with another protein hydrophobins to create a fusion protein


1. Molecular Cloning

For Molecular cloning, we selected pET28a plasmid. We successfully amplified four gene segments of MLCC (as control group), mLCC-linker-mHFBI, mLCC-linker-mHGBI and mLCC-linker-BslA. Then we digested and connected the four segments to pET28a vector through two restriction enzymes of BamHI and XhoI. At present, four recombinant plasmids have been successfully constructed.



(a)<img class="big-img" id="F23S" style="width : 500px;" src="T--BJEA_China--Resultbu.png">
(b)<img class="big-img" id="F23S" style="width : 500px;" src="T--BJEA_China--molecularcloning4.jpg">

(Fig1. (a). M: marker; Lane 1: mLCC 780bp; Lane 2: mLCC-linker-mHGFI 1059bp Lane 3: mLCC-linker-mHFBI 1035bp; Lane 4: mLCC-linker-BslA 1227bp
(b). M: marker; Lane 1,2,3,4: mLCC 780bp; Lane 5,6,7: mLCC-linker-mHGFI 1059bp Lane 8,9.10: mLCC-linker-mHFBI 1035bp; Lane 11,12,13: mLCC-linker-BslA 1227bp )



2. Protein Expression

We transformed four recombinant plasmids into BL21 expressing strains. For four recombinant strains, we tried three IPTG induction concentrations of 0.5mM, 0.7mM, 1.0mM and three induction times of 16h, 20h and 24h, respectively. We found that the induction concentration of 0.5mM IPTG and the induction time of 20h were the best.

(a)<img class="mid-img FLL" src="T--BJEA_China--mLCC.jpg">
(b)<img class="mid-img FLL" src="T--BJEA_China--clone2.jpg">
(c)<img class="mid-img FLL" src="T--BJEA_China--clone4.jpg">
(d)<img class="mid-img FLL" src="T--BJEA_China--clone3.jpg">

Fig.2. (a). pET28a-mLCC transformed into BL21 expressing strains.
(b). pET28a-mLCC-linker-mHGFI transformed into BL21 expressing strains.
(c). pET28a-mLCC-linker-mHFBI transformed into BL21 expressing strains.
(d). pET28a-mLCC-linker-BslA transformed into BL21 expressing strains.

 


(a)<img class="big-img" src="T--BJEA_China--%E8%9B%8B%E7%99%BD%E8%83%B61.jpg">
(b)<img class="big-img" src="T--BJEA_China--%E8%9B%8B%E7%99%BD%E8%83%B62.jpg">
(c)<img class="big-img" src="T--BJEA_China--%E8%9B%8B%E7%99%BD%E8%83%B63.jpg">

Fig.3. (a).Induction time:16h; M: marker; (b).Induction time:20h; M: marker;(c). Induction time: 24h; For all lanes 1-12 in (a), (b), (c):
Lane 1,2,3: pET28a-mLCC-mHFBI, induction concentration: 0.5mM, 0.7mM,1.0mM;
Lane 4, 5, 6: pET28a-mLCC-mHGFI, induction concentration: 0.5mM, 0.7mM,1.0mM;
Lane 7, 8, 9: pET28a-mLCC, induction concentration: 0.5mM, 0.7mM,1.0mM;
Lane 10, 11, 12: pET28a-mLCC-BsIA, induction concentration: 0.5mM, 0.7mM,1.0mM;

 



3. Protein Purification

(Because the pET28a plasmid has His-tag), therefore we used affinity chromatography to purify the supernatant obtained after clastogenesis using nickel. The result of our proteogels after elution with 200 mm imidazole and 300 mm imidazole is shown below. We successfully purified MLCC and MLCC-BslA. At the same time, we can also see that the two proteins, MLCC-mHGFI, MLCC-mHFBI, are expressed, but the protein expression is very low.

<img class="big-img" id="F23S" src="T--BJEA_China--%E8%9B%8B%E7%99%BD%E8%83%B64.jpg">

 

Fig.4. (a). M: marker;

Lane 1, 2: mLCC-linker-BslA 43kDa, elution concentration: 200mm Imidazole, 300mm Imidazole;
Lane 3, 4: mLCC 28kDa, elution concentration: 200mm Imidazole, 300mm Imidazole;
Lane 5, 6: mLCC-linker-mHFBI 36kDa, elution concentration: 200mm Imidazole, 300mm Imidazole;
Lane 7, 8: mLCC-linker-mHGFI 36kDa, elution concentration: 200mm Imidazole, 300mm Imidazole;





4. PET Degradation Reaction

We perform PET degradation reaction to detect enzyme activity. We set several protein concentration to detect enzyme activity, under the condition of pH8,70°C, and 18h of reaction time. Then we measured the absorption value at uv240nm by nanodrop, which is the absorption position of the product TPA, and we were surprised to find that the relative enzyme activity of mLCC-linker-BslA was increased about 3 times compared to mLCC!


(a)<img class="big-img" id="F23S" src="T--BJEA_China--Zhexiantu.png">
(b)<img class="big-img" id="F23S" src="T--BJEA_China--Zhuzhuangtu.png">

Fig. (a). The adsorption value of mLCC, mLCC-Linker-BslA of UV240 under the condition of pH8, 70 celsius degree, reaction time of 18h.
(b) The relativity activity of mLCC, mLCC-Linker-BslA.

 

 

Method 2: Cell surface display of mLCC in B-subtilis.


1. Molecular Cloning

During cloning we used pHT43 plasmid. We successfully performed PCR amplification of all the four fusion genes. As we continued our lab, recombinant plasmid with CotB was successfully constructed after the double digestion verification (pHT43-cotB-linker-mLCC). However, the other three CotC, CotG, and CotX are still continuing to be tried out.



(a)<img class="max-img" src="T--BJEA_China--BSmolecularcloning1.jpg">
(b)<img class="mid-img" src="T--BJEA_China--molecularcloning3.jpg">
(c)<img class="mid-img" src="T--BJEA_China--CotB.jpg">

 

Fig. 5. (a). M: marker; Lane 1: cotB-linker-mLCC; Lane 2: cotC-linker-mLCC; Lane 3: cotG-linker-mLCC 1426bp Lane 4: cotX-linker-mLCC 1357bp

(b). M: marker; Lane 1: cotB-linker-mLCC 1986bp ; Lane 2: pHT43-cotB-linker-mLCC (after construction);
(c). cotB-linker-mLCC transformed into pHT43 plasmid

Pre-expression:

The BL21 bacteria that contains aimed protein were cultured in 5mL LB liquid medium with kanamycin in 37℃ overnight. After taking samples, we transfer them into 1L LB medium with kanamycin.

Cultured in bottles:

After culturing in 37℃ in bottles, we used 0.5mM IPTG induced in 16℃ for 24 hours. Then, we used 200mM imidazole to eluting and get left 1st, 3rd, 5th, 7nd aimed protein, and we used 300mM imidazole to eluting the left 2nd, 4th, 6th, 8th aimed protein.

References

[1] Tournier, V. , Topham, C. M. , Gilles, A. , David, B. , & Marty, A. . (2020). An engineered pet depolymerase to break down and recycle plastic bottles. Nature, 580(7802), 216-219.

[2] Sulaiman S , You D J , Kanaya E , et al. Crystal Structure and Thermodynamic and Kinetic Stability of Metagenome-Derived LC-Cutinase[J]. Biochemistry, 2014, 53(11):1858-1869.