Coding

Part:BBa_K4189005

Designed by: Fanny Bonil   Group: iGEM22_Aix-Marseille   (2022-10-09)

NOTOC__ PE5

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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE 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.

contraption-2.jpg

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 PE5 :

Elution (PE) Droplet (PE) Elution (PP) Droplet (PP) Replica (PE)
Result C1. 16 780 C2. 17 292 C1. 25 173 C2. 930 C1. 4600 C2. 42 716 C1. 14 416 C2. 889 C1. 5518 C2. 2289
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 PE 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 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.

The fact that the diluted concentration have a higher signal than the C1 is maybe caused by a poor dilution or maybe the lower the concentration is, stronger the adherence will be.


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//proteindomain
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