Difference between revisions of "Team:Warwick/Results"
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<h3 class="text-center text-white"> <b> Synthetic Fatberg DNA Extraction </b> </h3> | <h3 class="text-center text-white"> <b> Synthetic Fatberg DNA Extraction </b> </h3> | ||
| − | <p>We had been thoroughly advised of the difficulties to extract fatberg DNA from Dr Pachebat (University of Aberystwyth ) and Dr Love (University of Exeter) and thus decided to construct a synthetic fatberg | + | <p>We had been thoroughly advised of the difficulties to extract fatberg DNA from Dr Pachebat (University of Aberystwyth ) and Dr Love (University of Exeter) and thus decided to construct a synthetic fatberg order to test our Qiagen PowerFaecal DNA extraction kit. The production of a synthetic fatberg was investigated and optimized to give reliable results, wetwipes fragments where even added to properly recreate a real fatberg!.After spiking 21 different synthetic fatberg compositions with <i> E. coli </i> cells and extracting the DNA we nano-dropped the samples and ran a gel on them to check for results, which sadly shows no success. </p> |
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<p class="aligncenter"> | <p class="aligncenter"> | ||
<img src="https://static.igem.org/mediawiki/2019/7/7d/T--Warwick--2019-FatbergGel.png" height="50%" width="50%"/> | <img src="https://static.igem.org/mediawiki/2019/7/7d/T--Warwick--2019-FatbergGel.png" height="50%" width="50%"/> | ||
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</p> | </p> | ||
| − | <p> Several species were found, particularly <i> | + | <p> Several species were found, particularly <i> Pseudomonas </i> as TliA (Thermostable lipase A, BBa_K258006) and candidate lipases from literature are from the same species (<i>Pseudomonas Flourescens</i>). Lipase sequences were searched for using known conserved domains and 22 lipases were found being produced by bacteria in the fatberg sample. Running these lipase sequences through BLAST showed similarities of 84 to 55% identity, for 21 of the sequences, suggesting that 21 lipases found had yet to be discovered. |
<p> Despite finding these new lipases, we continued cloning our candidate lipases due to: </p> | <p> Despite finding these new lipases, we continued cloning our candidate lipases due to: </p> | ||
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<p> The data below shows that we saw lipase activity when induced with IPTG compared to un-induced. </p> | <p> The data below shows that we saw lipase activity when induced with IPTG compared to un-induced. </p> | ||
| − | <p> We also measured the kinetic parameters of each lipase construct using the lysed and induced cells however, this was unsuccessful for CMLP and CALA, which showed inconsistent speeds | + | <p> We also measured the kinetic parameters of each lipase construct using the lysed and induced cells however, this was unsuccessful for CMLP and CALA, which showed inconsistent speeds we suspect this is most likely due to the fact that they catalysed the reverse reaction generating ester bonds rather breaking them. BSAL however, was characterised using the same method as TliA (see lab book) which produced the following graphs below. </p> |
Revision as of 18:03, 21 October 2019
Results
Synthetic Fatberg DNA Extraction
We had been thoroughly advised of the difficulties to extract fatberg DNA from Dr Pachebat (University of Aberystwyth ) and Dr Love (University of Exeter) and thus decided to construct a synthetic fatberg order to test our Qiagen PowerFaecal DNA extraction kit. The production of a synthetic fatberg was investigated and optimized to give reliable results, wetwipes fragments where even added to properly recreate a real fatberg!.After spiking 21 different synthetic fatberg compositions with E. coli cells and extracting the DNA we nano-dropped the samples and ran a gel on them to check for results, which sadly shows no success.
Fearing that the DNA samples were too low to be detected in our gels, we amplified the DNA using 16S RNA PCR and ran another gel on the products but were again, unsuccessful as seen by the gel below.
Did We Extract Actual Fatberg DNA?
After finally being cleared to obtain and work on an actual fatberg sample in a biosafety level 2 laboratory, we re-tested the Qiagen PowerFaecal DNA extraction kit on the sample although we had doubts due to the kit being unsuccessful on our previous fatberg model. Unsurprisingly, the DNA yields were low and as seen on the gel below, our extraction failed. This prompted us to get in touch with Dr Justin Pachebat for a sample of extracted DNA from the Whitechapel fatberg.
Sequencing The Largest Fatberg In The World
The sample of fatberg DNA given to us by Dr Pachebat was sequenced using the MinION from Oxford Nanopore where we found 22 lipases, 21 of which are not referenced in public databases. Several species were also identified which correlated to species mentioned in literature sources (insert these here lmao)
Fearing a similar situation to our previous DNA extraction gels, we nano-dropped our sample and amplified it using PCR to produce the following gel which shows DNA of several lengths, particularly in the 3kb range.
Over 12GB of sequence was found in the DNA sequence and further analysis revealed the presence of several bacterial species shown in the graph below
Several species were found, particularly Pseudomonas as TliA (Thermostable lipase A, BBa_K258006) and candidate lipases from literature are from the same species (Pseudomonas Flourescens). Lipase sequences were searched for using known conserved domains and 22 lipases were found being produced by bacteria in the fatberg sample. Running these lipase sequences through BLAST showed similarities of 84 to 55% identity, for 21 of the sequences, suggesting that 21 lipases found had yet to be discovered.
Despite finding these new lipases, we continued cloning our candidate lipases due to:
* Limited quantity of the DNA sample (we feared an unsuccessful PCR would degrade the sample)
* The DNA could code for a pathogenic factor that we would be unaware of
* A serious lack of time
Did We Clone The Lipases Successfully?
No and yes
We began our cloning strategy by obtaining a plasmid backbone from the Corre group, based at the University of Warwick. This backbone, named pJCC005, is used for cloning with both E. coli and Streptomyces cells. 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, removing all the Streptomyces -related genes. This process was harder and more time-consuming 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.
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 - a high fidelity enzyme - and MyTaq polymerase - a more robust enzyme - and take the cleanest PCR products forward, as shown in the gel below.
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 as seen as below.
Additionally, after testing the dead cells on our tributyrin agar (see design) we were surprised to see that no lipase activity was found. This led us to hypothesize that perhaps the accumulation of these lipases inside our cells was toxic. Interestingly, after deciding to clone in a non-functional version of our compost metagenome lipase precursor we were surprised to discover that we were able to successfully grow colonies of our transformed cells. This further suggested the accumulation of our functional lipases within our cells was toxic.
A New Strategy
To combat this, we thought about using secretion tags to export the lipases from our cells and prevent their accumulation, 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 below. The lipases used included Lipase A, chain A from Candida antarctica (CALA), an alkaliphilic lipase from Bacillus subtilis (BSAL), our compost metagenome lipase precursor (CMLP) and the Thermostable lipase A (TliA) from Pseudomonas fluorescens (please see our design page for justifications on this selection of lipases). This new backbone included a T7 promoter, enabling the induction of lipase expression with Isopropyl-β-D-thiogalactoside (IPTG). We decided to use this backbone following discussions with Dr. Love from Exeter University, who advised we engineer our bacteria such that they secrete our lipases in a controlled manner. This would not be possible if the lipases were under the control of a constitutive promoter like 'glpT'.
The gel below reveals the products obtained following cloning, miniprepping of our clones and performing a PCR using primers specific to each of our chosen lipases. After obtaining Sanger sequencing data of each of our clones, we were really pleased to discover that we successfully cloned in three of our lipases, as well as a fragment of TliA. Our cells also successfully grew as seen in the plates below. These results further supported the hypothesis that our bacterial cells were dying due to the constitutive expression and accumulation of our lipases. Our next step was to characterise the lipase activity of our engineered E. coli expression strains (BL21 star) using both a quantitative and qualitative assay of our own design.
Determining Lipase Function
To ensure our lipases worked, we first began with a qualitative test by measuring the absorbance rate of our extracted lipase enzymes using p-nitrophenol
The data below shows that we saw lipase activity when induced with IPTG compared to un-induced.
We also measured the kinetic parameters of each lipase construct using the lysed and induced cells however, this was unsuccessful for CMLP and CALA, which showed inconsistent speeds we suspect this is most likely due to the fact that they catalysed the reverse reaction generating ester bonds rather breaking them. BSAL however, was characterised using the same method as TliA (see lab book) which produced the following graphs below.
Testing The New Lipases
After using out model to pick the best oil concentration parameters to grow our transformed lipase bacteria in, we decided to use a MicrobeMeter from Humane Technologies to save more time. This experiment showed that the transformed bacteria did not follow a traditional growth curve but rather created peaks of activity. The doubling times below show how the plasmids affected the growth rate.
We repeated the experiment with the addition of IPTG 5 hours into growth to see the effect of the gene on the growth and found growth curves similar to the un-induced cells and similar doubling times as seen below.
There is remarkable similarity between induced and non-induced CMLP and CALA growth curves, leading us to believe that our constructs were being transcribed without the use of IPTG.
Do Lipases Limit The Growth Of Bacteria?
The bizarre growth curves led us to believe that maybe lipases were affecting a process present in only a single stage of the bacterial growth stages. To investigate the effect of lipases on cells in lag phase we inoculated plates and immediately induced with IPTG which gave us interesting results as the constructs grew unaffected by induction. We thus believe that a process during the log phase is causing cells to die upon lipase production.
What was also suspicious was the seemingly perfect coordination of the cell’s death between lipase constructs. This is highlighted in figure 1, where non induced CALA and CMLP constructs both reach the same optical density and then seemingly level off.
We hypothesized that our lipase was being induced by a process triggered by quorum sensing. Unfortunately, we were unable to further test this theory because of time constraints.
Summary
Therefore, from sequencing fatberg DNA to find lipase activity to use, to successfully cloning our constructs despite failing the first time round, we managed to find conclusive data, both qualitative and quantitative to suggest that our transformed bacteria are able to help degrade fatberg deposits, and to provide data to be used in the future of synthetic biology.
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