Part:BBa_K4189014
_NOTOC__ PP7
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 PP 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., Sq, K., and Wilkins, A.E. (54) POLYPROPYLENE BINDING PEPTIDES AND. 55.
Sequence and Features
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 2
- 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 PP7 :
| Elution (PP) | Droplet (PP) | Elution (PE) | Droplet (PE) | Replica (PP) | |
|---|---|---|---|---|---|
| Result | C1. 12 721 C2. 5995 | C1. 1293 C2. 433 | C1. 18 055 C2. 11 640 | C1. 8063 C2. 1682 | C1. 8119 C2. 895 |
| 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 put in contact with a PE plastic and Elution(PP) was put in contact with a PP plastic.
Elution PP has a stronger signal than SDS alone and GFP alone whether it is in the first concentration (350µg/µL) or at that diluted to 1/4. This means the protein has adhered correctly to PP plastic. On the other hand, the same result can be observed while in contact with PE plastic. Even if this protein adhere to PP plastic, there is no specific adherence capacity. The signals are higher with Elution (PE) than Elution(PP), so those results tend to lead us to say that this peptide adheres more to PE plastics than PP plastics.
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