Team:UESTC-China/Parts
We registered a total of 29 parts this year, including 21 basic parts and 8 composite parts, which are derived from 6 plasmids designed by us, and most of them have been tested to ensure their reliability. You can click hereto
learn more about our project to see their specific functions, or click on the part numbers when browsing the website.
Basic parts
Among the 21 basic parts, most are coding sequences that express enzyme proteins, including cellulase, xylanase, Lipase and Laccase, linkers like (GGGGS) linker, dockerin and cohesin domains. And some are sequences of common tags for detection.
| Part number | Name | Type | Function | Designer | Length |
|---|---|---|---|---|---|
| BBa_K3819003 | EGL7 | Coding | Cellulase EGL7, degrades cellulose. | Yuwei Hu | 1380 |
| BBa_K3819069 | EG1 | Plasmid | Cellulase EG1, degrades cellulose. | Yuanyi Wang | 1730 |
| BBa_K3819066 | CcsDockerin | Coding | Bind with CcsCohesin on the Scaffoldin. | Liyan Wu | 261 |
| BBa_K3819062 | XynB | Coding | Xylanase XynB, degrades lignin. | Liyan Wu | 675 |
| BBa_K3819072 | (GGGGS) 3 linker | Coding | Flexible linker, ligated XynB and CtDockerin. | Yuanyi Wang | 45 |
| BBa_K3819015 | CtDockerin | Coding | Bind with CtCohesin on the Scaffoldin. | Liyan Wu | 207 |
| BBa_K3819000 | CcsCohesin | Coding | Bind with CcsDockerin on the EG1 or EGL7. | Zhou Ying | 360 |
| BBa_K3819025 | Pro/Thr-rich Linker | Conjugation | Rigid linker, ligated the components on the Scaffoldin. | AllysonC | 108 |
| BBa_K3819004 | CBM3 | Coding | Cellulase binding module. | AllysonC | 477 |
| BBa_K3819006 | CtCohesin | Coding | Bind with CtDockerin on the XynB. | Zhou Ying | 429 |
| BBa_K3819008 | Type II dockerin | Coding | Bind with type Ⅱ cohesin on SdbA. | Zhou Ying | 486 |
| BBa_K3819050 | Myc-tag | Tag | As a tag for detection. | Luo SongWen | 30 |
| BBa_K3819147 | SdbA | Coding | Anchoring to the yeast cell surface. | Yujiao Peng | 1893 |
| BBa_K3819055 | Flag-Tag | Tag | As a tag for detection. | Liyan Wu | 24 |
| BBa_K3819005 | SMT3 | Other | Fusion expression tag, promotes protein expression. | Luo SongWen | 303 |
| BBa_K3819016 | (GGGGS) 2 linker | Coding | Flexible linker, ligated SMT3 and Lipase. | Luo SongWen | 30 |
| BBa_K3819010 | E2A | Coding | Ligand Laccase and Lipase. | Luo SongWen | 60 |
| BBa_K3819128 | Lipase | Coding | Lipase, degrades lignin. | Yujiao Peng | 891 |
| BBa_K3819009 | Gap Promoter | Regulatory | Promoter(no methanol induction required). | Yuwei Hu | 477 |
| BBa_K3819002 | Balac | Coding | Laccase, degradation of ink components. | Luo SongWen | 1740 |
| BBa_K3819051 | 6x His-tag | Tag | As a tag for Laccase detection. | Luo SongWen | 18 |
Composite parts
We registered 8 composite parts this year, these composite parts can express deinking enzymes and cellulosome components, which can ultimately assemble together to deink. BBa_K3819012 is our favorite one, combined different types cohesin and dockerin
to bind with corresponding domains to assemble into a cellulosome system, which can help inprove the efficiency of deinking.
| Part number | Name | Type | Function | Designer | Length |
|---|---|---|---|---|---|
| BBa_K3819077 | GAP Promoter--EGL7--(GGGGS)3 linker--CcsDockerin--6x His-tag | Composite | Express EGL7 with CcsDockerin to deink. | Liyan Wu | 2211 |
| BBa_K3819099 | EG1--(GGGGS)3 linker -linker-CcsDockerin--His-tag | Composite | Express EG1 with CcsDockerin to deink. | Liyan Wu | 1784 |
| BBa_K3819088 | XynB - GS linker - CtDockerin - His | Composite | Express XynB with CtDockerin to deink. | Liyan Wu | 969 |
| BBa_K3819033 | SdbA-FLAG | Composite | Express SdbA which can anchor to the yeast cell surface. | Liyan Wu | 1925 |
| BBa_K3819022 | Lipase plasmid | Plasmid | Express Lipase to deink. | Luo SongWen | 1956 |
| BBa_K3819021 | Balac plasmid | Plasmid | Express Laccase to deink. | Luo SongWen | 2291 |
| BBa_K3819023 | Lipase-Balac plasmid | Plasmid | Lipase and Laccase are designed in one plasmid to express. | Luo SongWen | 4387 |
| BBa_K3819012 | Self-design Scaffoldin | Composite | Express Scaffoldin binding with EG1 or EGL7 and XynB to deink. | Zhou Ying | 2435 |
Parts collection
We designed 6 plasmids this year. All of proteins expressed by these plasmids played a significant role in paper deinking process and they could assemble into our designed cellulosome system(see in Figure1.). That is why we make them a choice.
Cellulosome is a self-assembled multienzyme complex, which can enhance the targeting effect of cellulose through CBM domain, promote the spatial proximity of various enzymes and play a synergistic role. These advantages provide a good idea for developing
a method to improve the effective degradation of cellulose substrate. We can achieve higher deinking efficiency through cellulosome.
Figure1. Our self-design cellulosome system.
The core of the cellulosome design is the Scaffoldin protein containing a cellulose binding module(CBM) which binds cellulose substrates to enhance the targeted effect of enzymes. In addition, the most important structural domains of cellulosome are cohesin
and dockerin domains. Cohesin and dockerin are a pair of domains with specific interactions. And through this specific interaction, we could accurately control the proportion of enzymes by changing the number of different types of
cohesin domains. For example in Figure2., the proportion of EGL7 enzyme and XynB enzyme is controled to be 2:1, because the CcsCohesin: CtCohesin = 2:1.
Figure2. Interaction of cohesin and dockerin domain in cellulosome.
According to our experimental results, we tested that cellulase and xylanase were the best combination for our in-situ deinking without pulping, so we decided to assemble it on cellulosome. pEGL7 and pEG1 are designed to express EGL7 and EG1 cellulase
with CcsDockerin domains respectively. Cellulase can cut cellulose molecules connecting paper fiber and ink, prompting separation of ink from paper. Cellulase plays an important role in deinking. pXynB plasmid expresses xylanase with
CtDockerin domain and xylanase promotes the hydrolysis of hemicellulose in the fiber-ink binding region, which leads to ink separation.
Figure3. pEGL7, pEG1 and pXynB plasmids and their expressed proteins
pScaf plasmid expresses Scaffoldin, and there are two kinds of cohesins in it: Ccscohesin and Ctcohesin, whose proportion is 2:1. The cellulase and xylanase will maintain a 2:1 ratio through cellulosome. The assembly structure of cellulase, xylanase and
Scaffoldin is shown as follows:
Figure4. pScaf plasmid and Scaffoldin protein
SdbA makes cellulosome: Scaffoldin and enzymes docking on it, anchor together on Pichia pastoris. It may increase the stability of the overall enzyme system. SdbA protein can also be used as a tool to detect if the cellulosome has successfully assembled
or not. For example, SdbA makes cellulosome anchor on the surface of Pichia pastoris. If we treat the proteins of cellulosome system with their antibodies which has fluorescence, corresponding fluorescence can occur on the surface
of cells, which indicates that cellulosome complex is successfully assembled on the surface.（see in Figure5.）.
Figure5. Pichia pastoris treated with different antibodies under a fluorescence microscope.
pLipLac plasmid expressed Laccase and Lipase. Laccase dissolves and destroys lignin in pulp by catalyzing the oxidation of lignin, and removes ink binding to lignin in pulp. Lipase can degrade some lipid linked substances contained in plant ink, removing
the ink group from the fiber. These two enzymes are not attached to cellulosome for the consideration that their performances are not the best in our experiment. If enzymes from other species behave well, the optimal synergic combination
may change, so it is ok that add dockerin domains to both Lipase and Laccase and assemble them together on the cellulosome.
Figure6. pLipLac plasmid and Laccase and Lipase protein without dockerin domain.
| Plasmid | Part number | Name | Type | Function | Designer | Length |
|---|---|---|---|---|---|---|
| 1.pEGL7: pGAPZ_EGL7-GSlinker-CCsDock-His | BBa_K3819003 | EGL7 | Coding | Cellulase EGL7, degrades cellulose. | Yuwei Hu | 1380 |
| BBa_K3819072 | (GGGGS) 3 linker | Coding | Flexible linker, ligated EGL7 and CcsDockerin. | Yuanyi Wang | 45 | |
| BBa_K3819066 | CcsDockerin | Coding | Bind with CcsCohesin on the Scaffoldin. | Liyan Wu | 261 | |
| BBa_K3819051 | 6x His-tag | Tag | As a tag for detection. | Luo SongWen | 18 | |
| BBa_K3819077 | GAP Promoter--EGL7--(GGGGS)3 linker--CcsDockerin--6x His-tag | Composite | Express EGL7 with CcsDockerin to deink. | Liyan Wu | 2211 | |
| 2.pEG1: pGAPZ_EG1-Gslinker-CCsDock-His | BBa_K3819069 | EG1 | Plasmid | Cellulase EG1, degrades cellulose. | Yuanyi Wang | 1730 |
| BBa_K3819072 | (GGGGS) 3 linker | Coding | Flexible linker, ligated EG1 and CcsDockerin. | Yuanyi Wang | 45 | |
| BBa_K3819066 | CcsDockerin | Coding | Bind with CcsCohesin on the Scaffoldin. | Liyan Wu | 261 | |
| BBa_K3819051 | 6x His-tag | Tag | As a tag for detection. | Luo SongWen | 18 | |
| BBa_K3819099 | EG1--(GGGGS)3 linker -linker-CcsDockerin--His-tag | Composite | Express EG1 with CcsDockerin to deink. | Liyan Wu | 1784 | |
| 3.pXynB: pGAPZ_a-factor-xynB-GSlinker-CtDock-His | BBa_K3819062 | XynB | Coding | Xylanase XynB, degrades lignin. | Liyan Wu | 675 |
| BBa_K3819072 | (GGGGS) 3 linker | Coding | Flexible linker, ligated EGL7 and CcsDockerin. | Yuanyi Wang | 45 | |
| BBa_K3819015 | CtDockerin | Coding | Bind with CtCohesin on the Scaffoldin. | Liyan Wu | 207 | |
| BBa_K3819051 | 6x His-tag | Tag | As a tag for Laccase detection. | Luo SongWen | 18 | |
| BBa_K3819088 | XynB - GS linker - CtDockerin - His | Composite | Express XynB with CtDockerin to deink. | Liyan Wu | 969 | |
| 4.pScaf: pGAPZ_a-factor-Scaffoldin-Myc | BBa_K3819000 | CcsCohesin | Coding | Bind with CcsDockerin on the EG1 or EGL7. | Zhou Ying | 360 |
| BBa_K3819025 | Pro/Thr-rich Linker | Conjugation | Rigid linker, ligated the components on the Scaffoldin. | AllysonC | 108 | |
| BBa_K3819004 | CBM3 | Coding | Cellulase binding module. | AllysonC | 477 | |
| BBa_K3819006 | CtCohesin | Coding | Bind with CtDockerin on the XynB. | Zhou Ying | 429 | |
| BBa_K3819008 | Type II dockerin | Coding | Bind with type Ⅱ cohesin on SdbA. | Zhou Ying | 486 | |
| BBa_K3819050 | Myc-tag | Tag | As a tag for Lipase detection. | Luo SongWen | 30 | |
| BBa_K3819012 | Self-design Scaffoldin | Composite | Express Scaffoldin binding with EG1 or EGL7 and XynB to deink. | Zhou Ying | 2435 | |
| 5.pGAPZ_SdbA-FLAG | BBa_K3819147 | SdbA | Coding | Anchoring to the yeast cell surface. | Yujiao Peng | 1893 |
| BBa_K3819055 | Flag-Tag | Tag | As a tag for detection. | Liyan Wu | 24 | |
| BBa_K3819033 | SdbA-FLAG | Composite | Express SdbA which can anchor to the yeast cell surface. | Liyan Wu | 1925 | |
| 6.pLipLac: pGAPZ_a-factor-SMT3-linker-Lipase-Myc-E2A-a-factor-BaLac-His | BBa_K3819005 | SMT3 | Other | Fusion expression tag, promotes protein expression. | Luo SongWen | 303 |
| BBa_K3819016 | (GGGGS) 2 linker | Coding | Flexible linker, ligated SMT3 and Lipase. | Luo SongWen | 30 | |
| BBa_K3819128 | Lipase | Coding | Lipase, degrades lignin. | Yujiao Peng | 891 | |
| BBa_K3819050 | Myc-tag | Tag | As a tag for detection. | Luo SongWen | 30 | |
| BBa_K3819010 | E2A | Coding | Ligand Laccase and Lipase. | Luo SongWen | 60 | |
| BBa_K3819002 | Balac | Coding | Laccase, degradation of ink components. | Luo SongWen | 1740 | |
| BBa_K3819051 | 6x His-tag | Tag | As a tag for detection. | Luo SongWen | 18 | |
| BBa_K3819022 | Lipase plasmid | Plasmid | Express Lipase to deink. | Luo SongWen | 1956 | |
| BBa_K3819021 | Balac plasmid | Plasmid | Express laccase to deink. | Luo SongWen | 2291 | |
| BBa_K3819023 | Lipase-Balac plasmid | Plasmid | Lipase and Laccase are designed in one plasmid to express. | Luo SongWen | 4387 |
Prats contribution
Codon optimized has been done for yeast expression of previous parts (BBa_K2155004, BBa_K2155007, BBa_K2155005 and BBa_K1830009) since we use pichia pastoris as chassis organism to express the enzymes and cellulosome components, because eukaryotes can
post-translationally process proteins and yeast can increase expression. And we found that the sequence information of CcsDockerin (BBa_K2155004) may be wrong, so we corrected it. We also changed the locations of CBM and different
Cohesin domains on the Scaffoldin (BBa_K2155012), which reduces the spatial hindrances between adjacent domains. More details can been found in Improvement of an existing part.
| Part number | Name | Source | Contribution | Codes in the lab |
|---|---|---|---|---|
| BBa_K2155004 | CcsDockerin | iGEM16_NWPU | Codon optimized for yeast expression. Perfected the sequence information of CcsDockerin. | EGL7 and EG1 |
| BBa_K2155007 | CcsCohesin | iGEM16_NWPU | Codon optimized for yeast expression | Scaffoldin |
| BBa_K2155005 | CBM3 | iGEM16_NWPU | Codon optimized for yeast expression | Scaffoldin |
| BBa_K2155012 | Scaffoldin | iGEM16_NWPU | Changing the locations of CBM and different Cohesin domains reduces the spatial hindrances between adjacent domains. Perfected the sequence information of Scaffoldin. | Scaffoldin |
| BBa_K1830009 | CtDockerin | iGEM15_Concordia | Codon optimized for yeast expression | XynB |
Improvement of an existing part
BBa_K3819066: CcsDockerin
Sequence is derived from the iGEM website, by the previous team iGEM16_NWPU upload part library (BBa_K2155004). We blast this sequence, and find that it is derived from the
whole genome of Clostridium cellulovorans (CP002160.1) and ranges from 4108482-4108742. Transcription proceeds in the direction from downstream 4108742 to upstream 4108482.
But we find a problem that the sequence of CcsDockerin in NCBI is 4108479-4111682, which is different from Blast's result, so we end up using the sequence information displayed in NCBI. And we have done codon optimization for yeast expression as we chose
Pichia Pastoris to be the chassis this year.
Figure7. Gene squence alignment of CcsDockerin from iGEM16_NWPU and UESTC-China
Figure8. Amino acid squence alignment of CcsDockerin from iGEM16_NWPU and UESTC-China.
The two figures above show that the amino acid sequence of the two parts are the same but the gene sequence is not completely the same.
BBa_K3819012:Scaffoldin
When we want to use the Scaffoldin protein sequence of Team iGEM16_NWPU, we import it to SnapGene and find that there are lots of termination codons in this sequence(see in Figure9.). We checked the description of this part and redesigned the sequence
based on the similar sequence information provided by NCBI.
Figure9. Termination codons in BBa_K3819012.
We also improve BBa_K2155012 of iGEM16_NWPU by changing the position of CBM domain from the end to the middle, putting CcsCohesin domains and CtCohesin domain on both sides of CBM
domain, and using rigid linker (Pro/ THR-rich, BBa_K3819025 ). These improvements reduce the steric hindrance of adjacent domain proteins, thus reducing the
impact on their function and enabling them to function normally.
Futhermore, this year our chassis organism is Pichia pastoris GS115, so we have done codon optimization for yeast expression, and the sequence alignment result of scaffoldin gene from team iGEM16_NWPU and our team is as Figure10. In addition, the amino acid sequence alignment result of cellulosomal-scaffolding protein A [Acetivibrio thermocellus] and our self-design scaffoldin protein is shown as Figure11..
Figure10. Gene squence alignment of Scaffoldin from iGEM16_NWPU and UESTC-China.
Figure11. Amino acid sequence alignment of cellulosomal-scaffolding protein A [Acetivibrio thermocellus] and our self-design scaffoldin protein.
Finally, we successfully expressed the redesigned Scaffoldin, which means the sequences after codon optimization can be well expressed in yeast cells. The colony PCR and DotBlot results are as follows：
Figure12. Colony PCR results of recombined GS115(pScaf). The left arrow refers to specific primers' product(1901bp), and the right one refers to non-specific primers' product(3047bp).
Figure13. The Dot-Blot results of recombined GS115 fermentation broth. All samples were gained after fermentation in YPD for 96h. EGL7(His-Tag), EG1(His-Tag), XynB(His-Tag), Scaffoldin(Myc-Tag), Laccase(His-Tag) and Lipase(Myc-Tag)
were tested by adding 2ul fermentation supernatant on NC membrane and exposing 60s.
References
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