Team:Edinburgh OG/Parts

Parts

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Basic Parts Description

K2940000

Registry

Results

This part encodes a heavy-metal biosensor that produces the CotA laccase from B. subtilis in response to the presence of zinc in the medium. Zinc is a common component of textile effluent and is thus used as an indirect indicator of dye contamination. CotA is placed under the smtA promoter, which is controlled by the metallothionein repressor protein SmtB. Experimental results show that laccase production is induced by zinc at levels below that commonly found in textile effluent, showing the sensor is sensitive enough to produce laccase at industrially relevant concentrations.

K2940003

Registry

Results

Silk proteins are composed of long stretches of monomer repeats. Major Ampullate Spidroin (MaSp1) protein is a monomeric component of dragline spider silk. This BioBrick encodes a single monomer of MaSp1 from N. clavipes with a system in place to build monomer repeats using repeated digestion and ligation steps (head-to-tail multimerization). This allows a simple way to build long chains of MaSp1 repeats to produce the silk protein fiber. The BioBrick also contains a polyglutamine sequence which helps to solubilize the produced protein.

K2940005

Registry

Results

This part encodes a flavin-independent azoreductase fused to an N-terminal SpyCatcher tag. This tag allows the enzyme to be functionally displayed using the SpyTag-SpyCatcher system. This would allow the enzymes to be displayed on curli fibres outside the cell, where they would come in contact with and break down azo dye. The azoreductase enzyme cleaves the azo N=N bond which is characteristic of azo dyes and has been shown to be able to decolourise many different dyes. Using an enzyme without the need for cofactors simplifies the process and helps ensure that the dye-degrading system is less reliant on its environment.

K2940006

Registry

Results

This part encodes the super-bright GFP known as sfGFP (super folder GFP) fused to the SpyCatcher tag. This BioBrick was designed to be used as a control for the functional display of proteins on curli fibres produced by E coli.

K2940007

Registry

Results

Peroxidases are a class of general oxidative enzymes that can break down azo dyes. DyP is a family of dye-decolourising peroxidases that can break down dyes, particularly anthraquinone dyes, which are the second most common class of dye. This BioBrick encodes DyP from B. subtilis, which is a promising candidate for dye bioremediation. DyP is normally a secreted enzyme.

K2940010

Registry

Results

This part encodes the DyP peroxidase which was intended to be used to create ‘ghost shells’ of dead E. coli cells with the DyP peroxidase anchored to the inner cell membrane. In order to achieve this anchoring, the signal peptide of DyP has been removed and replaced with the C-terminal of Cytochrome b5, which is a membrane anchor protein. In concert with a lysis protein, this was designed so the DyP peroxidase would become anchored to the inside of the membrane, and then cells would be killed, allowing free passage of dye into the cell shell, where it could be broken down. This would both avoid any negative effects on the living cell of the toxic breakdown products of dye, and provide a safety mechanism to avoid the release of living, genetically modified organisms.

Improved Part Description

K2940002

Registry

Results

This part encodes an improved version of the Bpul laccase first submitted as part BBa_K863000 by Bielefeld University in 2012. This is a laccase from B. pumilis which is a CotA-type laccase and has shown dye-degrading potential.We performed directed evolution via error-prone PCR to generate a library of laccase enzymes and tested them via ABTS assays. The sequence of this part contains eight beneficial mutations which improved the activity of the laccase over the wild type version.

Composite Parts Description

K2940001

Registry

Results

In this part, the PelB leader peptide (BBa_J32015) has been fused in front of Bpul laccase (BBa_K863000). PelB directs the unfolded fusion protein to the periplasm, where the peptide is cleaved by a signal peptidase and the protein secreted into the periplasm. The SHSBNU China iGEM team from 2018 used this as a strategy to increase the secretion of enzyme (here Bpul) into the extracellular environment, in order to avoid toxic substances needing to be taken into the cell to be detoxified. However, in this case, we did not find any appreciable extracellular secretion of Bpul by the addition of PelB.

K2940014

Registry

Results

This part encodes XylR:sfGFP biosensor genetic circuit, which was meant to serve as a platform for the directed evolution of a biosensor capable of detecting aromatic amines – the intermediates of azo dyes. In contrast to the original XylR:GFP biosensor version submitted as part BBa_K2023015 by Ionis Paris iGEM team 2016, the use of codon-optimized superfolder GFP as the reporter resulted in a nonfunctional biosensor exhibiting high uninduced sfGFP fluorescence levels and being otherwise not sensitive enough to toluene induction.

Part Characterisation

For our BioBrick characterisation, we chose to characterise three parts containing ribosomal binding sites under a medium constitutive promoter originally described by the Freiburg 2011 iGEM team (Part:BBa_K608010, Part:BBa_K608011,Part:BBa_K608012).

Summary

We compared the amount of GFP expressed under the medium constitutive promoter with strong, medium and weak RBS/GFP (PR4-GFP, PR5-GFP, PR6-GFP) in TOP10, BL21 (DE3) and DH5α cells lines. We were interested in replicating the original characterisation as the Freiburg 2011 team mentioned it needed to be replicated. We did so with 3 biological replicates and 3 technical replicates for each control. Additionally, we characterised these biobricks across different common E. coli strains to investigate the consistency across these strains.

Methods

Biobricks were extracted and amplified from the iGEM kit 2019 following standard protocols. Transformation was performed following heat-shock standard protocols. Three positive colonies were picked containing each biobrick in each different cell line. The samples were repeated in series of three to ensure consistency. This was carried out by adding 200 μl of cell culture to wells of a 96-well microplate. The plate reader was set at 485nm wavelength and 520nm emission for analysis.

Results

The next graph shows the average of the three samples (repeated three times) for each biobrick (PR4-GFP, PR5-GFP, and PR6-GFP) in different strains with their respective GFP fluorescence intensity. The weakest RBS (PR6-GFP) in BL21 presents the highest GFP absorbance, in contrast with the Freiburg data showing that the medium RBS (PR5-GFP) had the highest GFP absorbance.

Figure 1. GFP Flourescence Activity (PR4-GFP, PR5-GFP, and PR6-GFP)

Our data suggests that in BL21 the highest GFP expression was led by the weakest RBS (PR6-GFP), followed by the strongest RBS (PR4-GFP), and finally the medium RBS (PR5-GFP).

TOP10 shows the highest GFP expression with the strongest RBS (PR4-GFP), followed by the medium RBS (PR5-GFP), and finally the weakest RBS (PR6-GFP).

DH5a shows the highest GFP expression with the strongest RBS (PR4-GFP), followed by the weakest RBS (PR6-GFP), and finally the medium RBS (PR5-GFP).

TOP10 is the only cell line showing consistency in the hierarchy of the strongest to the weakest RBS.

Contact Us

Edinburgh OG
Peter Wilson Building
University of Edinburgh
edigemmsc@ed.ac.uk