Difference between revisions of "Part:BBa K1921015"

Latest revision as of 13:02, 12 October 2023

INPNC

Sequence and Features

Usage

This part is the fusion of N-terminal and C-terminal domain of the ice nucleation protein. Here we established an approach to display PETase on the surface of Escherichia coli (E. coli) using N-terminal and C-terminal of ice nucleation protein as anchoring motif. Bacteria cell surface display means we fix the enzyme onto the out membrane of E.coli. According to the immobilization the enzyme are capable to stay at a proper orientation so that they get more possibilities to combine with the PET. Because the highly hydrophilic C-terminal of INP can combine with the out membrane, the display of our passenger protein, PETase, are allowed to be more stable.Besides, our method solve the problem of the degradation PETase. The enzyme will be stable in the cell surface display system.

Biology

Surface expression of recombinant proteins was first described more than 30 years ago.INP is an OMP that is found in several plant pathogenic bacteria. Our inaK is from Pseudomonas. INP has several unique structural and functional features that make it highly suitable for use in a bacterial surface display system. The specific amino acids of the N-terminal domain are relatively hydrophobic and link the protein to the OM via a glycosylphosphatidylinositol anchor. The C-terminal domain of the protein is highly hydrophilic and exposed to the medium. The central part of INP comprises a series of repeating domains that act as templates for ice crystal formation.

Reference

[1] Shosuke, Yoshida, 1, 2*, Kazumi, Hiraga, 1, Toshihiko, Takehana, 3, Ikuo, Taniguchi, 4, Hironao, Yamaji, 1, Yasuhito, Maeda, 5, Kiyotsuna, Toyohara, 5, Kenji, Miyamoto, 2†, Yoshiharu, Kimura, 4, Kohei, Oda1. A bacterium that degrades and assimilates poly(ethylene terephthalate)[J]. SCIENCE, 2016: 1196-1199

[2]Edwin, van, Bloois1, Remko, T, Winter1, Harald, Kolmar2, and, Marco, W, Fraaije. Decorating microbes: surface display of proteins on Escherichia coli[J]. CELL Press, 2011, 29(2): 79-86

Additional Supplements

The infomation below is updated by TJU_China of iGEM 2017.

Characterization Description

Last year, this part did not submit the corresponding experimental data, we chose this part this year as part of the construction in suface display, while we cannot sure its characteerization. So we did two construction for surface display. One was for E.coli BL21, the other was for Citrobacter rodentium. There is no infomation on anchoring protein for C.rodentium, so we want to test this one (since these two strains both belong to Enterobacteriaceae). We used pET28b (with T7 promoter) in E.coli and pACYC184 (a low copy number plasmid which is repeatedly used in C.rodentium) in C.rodentium (with another promoter gathered from C.rodentium genome Part:BBa_K2328012).

We did western blot experiment for these two kinds of construction. The results are as shown in the figure. We can see that we can get our target protein (INPNC + smURFP, Part:BBa_K2328023) in lane 4, but nothing in the lane 2. It may indicated that this anchoring protein should be induced to be over-expressed to get a more satisfying effect. Or it just indicated that the promoter of C,rodentium or the low copy number plasmid were just not suitable for this anchoring protein.

Figure 1. The result of western blot. Lane 1 is C.rodentium with no plasmid. Lane 2 is C.rodentium with pACYC184. Lane 3 is E.coli BL21 with no plasmid. Lane 4 is E.coli BL21 with pET28b. Lane 5 is positive control (a protein with His-tag).

Improvements

Group: BJEA-China 2023

Author: Chenzhang Ma

A study published in Nature on October 20, 2022, by a team of researchers from Peking University, the National Institutes of Health (NIH) led by Frank Gonzalez, the First Affiliated Hospital of Zhejiang University School of Medicine led by Chaohui Yu, Fudan University School of Basic Medical Sciences led by Yang Li, and the First Affiliated Hospital of Wenzhou Medical University led by Minghua Zheng, explored the process. They found that nicotine, ingested during smoking, accumulates in the gut and accelerates the progression of NAFLD. Notably, they discovered that nicotine can be efficiently degraded by the human gut commensal bacterium B. xylanisolvens.

To identify the key enzyme responsible for nicotine degradation, the research team utilized chromatographic and spectroscopic techniques and identified the nicotine metabolite as 4-hydroxy-1-(3-pyridinyl)-1-butanone (HPB), which significantly differs from host nicotine metabolism products, representing a novel nicotine metabolite. Subsequently, through whole-genome sequencing and functional gene analysis, the team identified the potential nicotine degradation enzyme NicX in B. xylanisolvens, and in vitro enzymatic assays confirmed NicX's role in nicotine degradation.

Cell surface display system involves fusing the target protein with an anchor protein to express the protein on the cell surface. In this project, we are utilizing E. coli surface display system, fusing NicX with four different anchor proteins. Tianjin University established the parts anchoring protein INPNC (BBa_K1921015) in 2016, and in 2023, we used INPNC to display NicX on the cell surface. This system allows our fusion protein to degrade nicotine in the environment without purification.

In conclusion, our research efforts are driven by the practical goal of contributing to the degradation of nicotine, thereby playing a key role in environmental protection and human health.

Figure 1. (a) SDS-PAGE of INPNC- NicX- histag(1989bp) & NicX(1293bp) transformed into BL21 expressing strains. Induction time: 15h M:GoldBand Plus 3-color Regular Range Protein Marker(8-180 kDa), 1:INPNC- NicX- histag(1989bp)Before induction 2, 3, 4, 5, 6:After induction; 2: 37℃ 0.3mM IPTG,3: 37℃ 0.5mM IPTG,4: 37℃ 0.7mM IPTG,5: 37℃ 1mM IPTG 6: NicX(1293bp) Before induction 7,8:After induction; 7: 37℃ 0.3mM IPTG,8: 37℃ 0.5mM IPTG (b) 1: 37℃ INPNC- NicX- histag(1989bp)Before induction 2-6:After induction; 2: 37℃ 0.3mM IPTG,3: 37℃ 0.5mM IPTG,4: 37℃ 0.7mM IPTG,5: 37℃ 1mM IPTG；6: 37℃ NicX(1293bp) Before induction 7-8:After induction; 7: 37℃ 0.3mM IPTG,8: 37℃ 0.5mM IPTG

Figure 2. (a) SDS-PAGE of INPNC- NicX-histag(1989bp) & NicX(1293bp) transformed into BL21 expressing strains. Induction time: 15h M:GoldBand Plus 3-color Regular Range Protein Marker(8-180 kDa), 3: NicX (1293bp) Before induction 1,2: After induction; 1: 37℃ 0.5mM IPTG, 2: 37℃ 0.3mM IPTG 8:INPNC- NicX- histag(1989bp) Before induction 4,5,6,7:After induction; 4: 37℃ 1mM IPTG, 5: 37℃ 0.7mM IPTG, 6: 37℃ 0.5mM IPTG,7: 37℃ 0.3mM IPTG(b)3: 37℃ NicX (1293bp) Before induction 1-2: After induction; 1: 37℃ 0.5mM IPTG, 2: 37℃ 0.3mM IPTG 8: 37℃ INPNC- NicX- histag(1989bp) Before induction 4-7:After induction; 4: 37℃ 1mM IPTG, 5: 37℃ 0.7mM IPTG, 6: 37℃ 0.5mM IPTG,7: 37℃ 0.3mM IPTG

Figure 10. LC-MS experiment conditions 1

Figure 11. LC-MS experiment conditions 2

Figure 3. INPNC-NicX 1000x 250μM 0.5

Figure 4. INPNC-NicX 1000x 250μM 0.5

For INPNC-NicX, we observed a suspicious anomalous peak in the 0.5-hour chromatogram, specifically due to column residue. The reason for this occurrence is likely improper handling of bacteria during pretreatment. However, due to the presence of residue, the final enzyme activity should be greater than 0.002 μmol/min. When we determined the concentration of INPNC-NicX at OD600 = 1.6, based on previous experiments and empirical data, we estimated the cell density to be approximately 1.6 x 10^8 cells per milliliter, which becomes 1.6 x 10^5 cells per milliliter after a 1000-fold dilution. In the system, we added 2 μL of bacterial culture, corresponding to 320 cells. Based on the previous calculation, we know that the reaction rate for 10 ng of protein is around 0.039 μmol/min. Using this information, we can infer that cells containing about 0.5 ng of protein are effectively participating in the reaction. Therefore, we can conclude that there is approximately 0.16 ng of protein per hundred cells. Additionally, we already know that 320 cells correspond to 0.5 ng of protein and a reaction rate of 0.002 μmol/min. Scaling this up, 6,400 cells correspond to 10 ng of protein and a reaction rate of around 0.04 μmol/min, consistent with the wild-type. Furthermore, in practical terms, when cultivating 1 L of medium to an OD600 of 1, there are approximately 10^8 cells, equivalent to about 16,000 ng of protein or a protein concentration of around 16 mg/mL. Additionally, surface display technology does not require cell disruption and purification, making it far more efficient than traditional intracellular expression methods.