Part:BBa_K4189004
NOTOC__ PE4
Contents
Introduction
As we all know, plastic is not biodegradable. Indeed, it’s created from fossils such as crude oil and heated to create strong polymers that cannot be found in nature which results in micro-organisms that didn’t evolve to attack these strong and high energy-demanding bonds. That is why we tried to find and modify adherence proteins that bind our degradation system to the chosen plastics.
Usage and Biology
- it’s one peptide that is normally capable of binding to a PE plastic
The patent in which we found the peptides is the following one: Cunningham, S.D., Ford, C., Lowe, D.J., O’Brien, J.P., Wang, H., Square, K., and Wilkins, A.E. (54) POLYETHYLENE BINDING PEPTIDES AND METHODS OF USE. 55
Sequence and Features
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Characterization
The promoter was characterized using GFP to measure gene expression at different concentrations and on various plastics to check its affinity and specificity. To do so, we inoculated E. Coli BL21 strain with pET GFP in LB-medium, and after inoculation, we induced and purified it in an Econocolumn Biorad.
The GFP was used as a tracker to be able to, during our adhesion tests to quantify the protein that adhered to the plastic by using a fluorometer.
Adherence tests
We had some issues finding and creating the proper protocol since we wanted consistent qualitative, and quantitative results. To start, we used the test of another iGEM team as a base to then modify it for our own tests: https://static.igem.org/mediawiki/2019/c/c8/T--Kyoto--wikimethod.pdf
They created the following part of it: Part: BBa_K3185007
First, we created a little contraption where our solution was put on glass tubes upside-down during a set time to keep the surface contact size uniform for each and every test.
Then, we decided to use 50 μL droplets, creating a perfect dome on the desired plastic. |400px|Figure 1. 50 μL droplets
We also prepared a dilution at ¼ of the solution to know to what extent the concentration helps the adherence. We then used a Tecan, a Fluorescence Microplate Reader, to quantify the GFP left on the plastic after three washes.
Results
Fluorometer result for PE4 :
| Elution (PE) | Droplet (PE) | Elution (PP) | Droplet (PP) | Replica (PE) | |
|---|---|---|---|---|---|
| Result | C1. 31 333 C2. 9824 | C1. 4884 C2. 781 | C1. 6556 C2. 24168 | C1. 1601 C2. 693 | C1. 9650 C2. 5842 |
| SDS alone | C1 3441 | C1 3408 | C1 3902 | C1. 2816 | C1. 2000 |
| GFP alone | C1. 4428 C2. 2640 | C1. 27549 C2. 523 | C1. 11 800 C2. 4288 | C1. 1130 C2. 317 | C1. 3998 C2. 3997 |
Elution(PE) means that the protein was in contact with a PE plastic, and Elution(PP) was in contact with a PP plastic.
Elution PE has a stronger signal than SDS alone and GFP alone, whether in the first concentration (350µg/µL) or at that diluted to 1/4. This means the protein has adhered correctly to PE plastic. On the other hand, GFP has a stronger signal than the protein when it’s in contact with PP plastic, meaning that this protein is very specific to PE.
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