Part:BBa_K4995002
Truncated ice nucleation protein+linker+cellulose binding protein
We found 2022 Vilnius-Lithuania igem team representation of a cellulose binding protein (CBD, https://parts.igem.org/Part:BBa_K4380000), We decided to use part of it as a binding protein for a microbial fixation technology based on bacterial cellulose (Figure 1). We also received the selfless help of the team at Squirrel-Beijing-I, who donated their bacterial cellulose to help us verify the feasibility of their fixation technology.
Usage and Biology
Figure 1 Protein structure of CBD（This image derived from Part:BBa_K4380000，pictured by 2022 Vilnius-Lithuania).
Figure 2 Design of gene circuit of INP-PETase and INP-CBD co-expression system.
Characterization
The adhesion effect of the recombinant strain to bacterial cellulose was tested. The sterile bacterial cellulose membrane (BC membrane) was cut into 5 cm x 5 cm pieces. The recombinant E. coli was cultured overnight in 5 mL LB medium containing 100 μg/mL ampicillin. The following day, bacterial pellets were collected by centrifugation, washed with PBS (pH 7.4), and the recombinant E. coli was resuspended to adjust the bacterial OD600 = 0.5. The recombinant E. coli suspension was then incubated with the BC membrane for 1 hour. Afterwards, the membrane was gently rinsed with 10 mL PBS to remove non-specifically adhered cells, and the rinse solution was collected. The rinsing solution was subsequently diluted in a gradient and spread onto LB AGAR plates containing ampicillin.. For comparison, wild-type E. coli was processed in the same way. After 24 hours of incubation, we calculated the CFU/mL for each sample. By comparing the non-specific adhesion of the INP-CBD fusion protein E. coli with that of the wild type on the bacterial cellulose membrane, we evaluated the adhesive effect of INP-CBD.
Figure 3 A: Explore the adhesion of CBD enhancement to cellulose membrane; B: Difference analysis of experimental results；C：Agarose gel electrophoresis image of INP-linker-CBD.
After the recombinant Escherichia coli strain was incubated with bacterial cellulose membrane, the CFU value of the washing solution was counted. The wild type strains without INP-CBD fusion had the least adhesion to bacterial cellulose membrane. The average CFU of flushing solution is 1.2 × 10^6 CFU/mL. The average CFU of the rinse solution of recombinant Escherichia coli strain was 7.6 × 10 ^ 5 CFU/mL (Figure 3). T-test was used for comparison. The p values obtained were all less than 0.05, indicating that there was a statistically significant difference in the adhesion ability of the two strains. This significantly enhanced adhesion confirmed the successful display of CBD on the surface of Escherichia coli and its function of binding to cellulose membrane.
Potential application directions
Figure 4 Illustration of hardware for water microplastic treatment.
We have successfully characterized the ability of INP-CBD, which effectively promotes the adhesion of Escherichia coli to bacterial cellulose. This achievement forms a robust foundation for the immobilization of our engineered bacterial strains. These immobilized engineered strains have the potential to be deployed in open water environments for pollution remediation. In our research, we will employ INP-PETase to address microplastics found in sewage.
We have established that INP-CBD can facilitate the adhesion of Escherichia coli to bacterial cellulose, providing us with an efficient means to firmly immobilize our engineered bacterial strains onto specific carriers. These immobilized engineered strains will serve as potent tools for tackling various types of pollutants in environmental settings.
These immobilized engineered strains hold promise for application in open water bodies to address a range of pollutants, including but not limited to organic waste and microplastics. In our study, we will specifically focus on the utilization of INP-PETase to address microplastics present in wastewater.
Microplastics represent a significant environmental concern, as they are widespread in natural water bodies and have adverse effects on aquatic life and the entire ecosystem. By harnessing our engineered strains, particularly those carrying variants of INP-PETase, we aim to explore a novel approach to efficiently degrade and remove these microplastic particles, mitigating their environmental impact.
Our research not only aims to address the microplastics issue but also paves the way for broader studies in environmental pollution control. This achievement has the potential to offer innovative solutions for creating a cleaner and healthier environment, highlighting the significance and potential of biotechnology in environmental conservation. We look forward to future experiments and research to validate the feasibility and effectiveness of our approach in real-world applications.
References
Dou, Jian-lin, et al. "Surface display of domain III of Japanese encephalitis virus E protein on Salmonella typhimurium by using an ice nucleation protein." Virologica Sinica 26 (2011): 409-417. Morag, Ely, et al. "Expression, purification, and characterization of the cellulose-binding domain of the scaffoldin subunit from the cellulosome of Clostridium thermocellum." Applied and environmental microbiology 61.5 (1995): 1980-1986.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 72
Illegal NgoMIV site found at 405 - 1000COMPATIBLE WITH RFC[1000]
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