Team:Warwick/Design

Project Design

Our idea was to utilise lipase activity in bacteria to use them to degrade fatbergs, but in order to do this, we had to understand various aspects of our idea, from choosing lipases, sequencing an actual fatberg, to finding methods to quantify and test for lipid degradation, we definitely had our hands full!

Lipase Cloning

Whilst we were waiting for both our fatberg sample from United Utilities and fatberg DNA provided by Dr. Justin Pachebat, we searched various databases and literature sources for other candidate lipases to clone into our E. coli cells.

Our starting point for this was the Thermostable lipase A (TliA) derived from the bacterial species Pseudomonas fluorescens - an iGEM part previously used by other teams such as Sheffield, Stuttgart and KAIST. BLAST searching TliA on the NCBI database revealed some similar lipases which were considered as potential candidates for our cloning strategy, including a lipase precursor from a compost metagenome.

Other lipases and lipase-producing species were identified from research papers investigating industrial wastewater treatment plants, lipid-rich wastewater and restaurant wastewater. The complete list of selected lipases can be viewed in figure 1

Backbones -pJCC005

Following on from our lipase selection, we obtained our starting backbone pJCC005, which was provided to us by the Corre group based at the University of Warwick. This plasmid is used with both Streptomyces and E. coli strains.

As such, this plasmid contained a large portion of Streptomyces related genes (labelled “to delete” in figure 2 below) that were irrelevant to our work. Consequently, we decided to design primers to amplify the part of the backbone we needed to create a new backbone optimised for transformation into our E. coli cells

This process was harder than anticipated due to the size of the pJCC005 backbone, requiring us to amplify the backbone in two parts and ligate them back together via Gibson assembly. Despite the challenge we were able to successfully make our own, new backbone pJC_BB12 (figure 3).

Insertion & Fusion

pJC_BB12 has a glpT promoter for constitutive expression of our lipases and contains a gene encoding the super-folded green fluorescent protein (sfGFP), which conveniently made our bacterial colonies fluoresce green, facilitating our selection process of cells containing our lipases. We also hoped the fluorescent property of sfGFP would allow us to track both the expression and movement of our lipases.

We had two strategies for cloning our lipases into our backbone. Firstly, at the N-terminus of sfGFP with each of our lipases possessing a ribosome binding site (RBS), as well as a start and stop codon to produce two separate proteins - a lipase and sfGFP.

We also wanted to fuse our lipases to the N-terminus of sfGFP to create one green fluorescent protein. In this scenario, both the lipase and sfGFP genes share an RBS, stop and start codons. In order to achieve this, we had to amplify our lipases in two slightly different ways for Gibson assembly. We decided to do this using both a Phusion polymerase and MyTaq polymerase and take the cleanest PCR products.

Changing Plasmids

We were really pleased to find we had successfully amplified seven of our eight lipases for both insertion next to the N-terminus of sfGFP and fusion at the N-terminus of sfGFP. However, we were a little baffled when we plated our transformed E. coli cells and incubated them only to discover that nothing grew on any of our plates.

We repeated our transformation a few times and to our amazement the same result kept repeating itself. Interestingly, when we cloned in a non-functional version of our compost metagenome lipase precursor we were able to successfully grow colonies of our transformed cells.

This led us to hypothesize that perhaps the accumulation of these lipases inside our cells was toxic. To combat this, we thought about using secretion tags to export the lipases from our cells, as well as putting our lipases under the control of an inducible promoter. Consequently, our next strategy involved cloning a small selection of our candidate lipases (since at this point we were pressed for time) into a new vector: pET151/D-TOPO, as shown in figure 5 below.

The lipases used included Lipase A, chain A from Candida antarctica, an alkaliphilic lipase from Bacillus subtilis, our compost metagenome lipase precursor and the Thermostable lipase A from Pseudomonas fluorescens. This new backbone included a T7 promoter, enabling the induction of lipase expression with Isopropyl-β-D-thiogalactoside (IPTG).

Assays

We then designed quantifiable assays to ensure we could measure the effect of our lipase transformed bacteria. We looked into using tributyrin agar plates, which are blue, and form colourless halos upon lipase activity of transformed bacteria

We also looked at using p-nitrophenol octanoate which is catalysed to p-nitrophenol and octanoic acid by Thermostable Lipase A (TliA) to produce a yellow colour change. We used this experiment to characterise TliA as a bronze requirement and then used this to measure the activity of our transformed candidate lipases which we quantified using photo-spectrometry.