Part:BBa_K4189011

_NOTOC__ PP4

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 3
  
• 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.

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 PP4 :

	Elution (PP)	Droplet (PP)	Elution (PE)	Droplet (PE)	Replica (PP)
Result	C1. 2908 C2. 1868	C1; 15704 C2. 1537	C1. 1843 C2. 2057	C1. 29 975 C2. 2935	X
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. GFP has a stronger signal than the Elution(PP), meaning there is no adhesion to PP plastic. The protein in contact with PE plastic has a lower signal than SDS alone and GFP alone whether it is in the first concentration (100µg/µL) or at that diluted to 1/4, so it can't adhere to PE plastic neither. This means the protein does not adhere to PE plastic yet we can't say that there is a specificity of adherence.