Difference between revisions of "Part:BBa K3039019"

Revision as of 17:01, 21 October 2019

PETase Reconstructed Ancestor 3

An important part of our project is engineering more stable enzymes that will last longer in our filter. In order to achieve this, we decided to make use of the method of ancestral reconstruction. This technique is used to recover ancestral traits that are useful but have been lost during the process of evolution; and relies on a large number of sequences, phylogenetic analysis, and modelling.
We started by searching current papers for phylogenetic trees of PETase that have already been built. We identified a paper that had already organised plastic-degrading enzymes into categories, and had identified a set of enzymes that were closely related to PETase. We used BLAST software to search for sequences homologous to PETase and the set of enzymes previously identified in the paper; and through this process identified 243 homologous sequences. These sequences were aligned and narrowed down to 76 sequences with the help of Professor Harmer from the University of Exeter Living Systems Institute. The final alignment of the sequences was then fed into the ANCESCON software that performed the ancestral reconstruction. The software produced a phylogenetic tree and identified 74 ancestors. In order to identify the most suitable ancestors to model and use, we used the method below to weight each one:
Following this method, we identified 4 potentially suitable ancestors to further analyse. The YASARA software was used to model the 3D structure of each of the four ancestors. The models produced were then aligned against the structure of PETase by Professor Harmer in order to identify significant changes in the sequences. We discovered that the catalytic triad was conserved in all four ancestors, suggesting that the PET degrading activity had not been lost. Interestingly, we have also discovered that a beneficial mutations reported in past papers was already present in all four of the ancestors, namely R280A. The only significant trait lost during the reconstruction was the second disulfide bond that was present in PETase but not the ancestors. However, we reverted this by changing the two alanine residues in the ancestors with two cysteine residues. Additionally, we deleted the first five amino acids from the N-terminus that we suspected were composing the signal peptide. Once these minor adjustments had been completed, we sent the final sequences to be synthesised.

Characterisation

In order to characterise our part and determine the rate of its activity and prove its functionality we have run a series of experiments. After transforming the Arctic Express, Rosetta Gami and BL21 DE3 strains of E. coli with our plasmid we induced the expression of the enzymes using IPTG. In order to confirm that the enzyme expression has been successful we ran a western blot which showed the presence of the enzyme in the soluble fractions of the sonicated cells. Afterwards the enzyme was purified and used in assays to show its functionality and determine the rate of its activity.

Expression in E.coli

Protein Purification

Assays

Esterase Assays

Thermal Stability Assay

Thermal Shift Assay

PET Assay

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 255
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 255
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 255
Illegal BamHI site found at 21
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 255
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 255
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K3039019 short</partinfo>
-The enzymes PETase and MHETase were first discovered in Ideonella sakaiensis in 2016 by a group of researchers in Japan. These enzymes were found to degrade polyethylene terephthalate (PET) into its monomers, terephthalic acid (TPA) and ethylene glycol (EG). PETase degrades PET into Mono-(2-hydroxyethyl)terephthalic acid (MHET), Bis(2-Hydroxyethyl) terephthalate (BHET) and TPA, the main product being MHET. MHET is further degraded by MHETase into TPA and EG. We are aiming to use mutants of these enzymes to degrade the microfibres that are coming off clothing during washing cycles. The enzymes would be secreted into a filter that captures the microfibres. This is the sequence of one of the four reconstructed ancestors of PETase with a His tag attached to it. The sequence has been obtained through the method of ancestral reconstruction. The His tag has been used in order to more easily identify the enzyme.
+<p>An important part of our project is engineering more stable enzymes that will last longer in our filter. In order to achieve this, we decided to make use of the method of ancestral reconstruction. This technique is used to <b><span class="blueText">recover ancestral traits that are useful but have been lost during the process of evolution;</span></b> and relies on a large number of sequences, phylogenetic analysis, and modelling.
+<br />
+We started by searching current papers for phylogenetic trees of PETase that have already been built. We identified a paper that had already organised plastic-degrading enzymes into categories, and had identified a set of enzymes that were closely related to PETase. We used BLAST software to search for sequences homologous to PETase and the set of enzymes previously identified in the paper; and through this process identified 243 homologous sequences. These sequences were aligned and narrowed down to 76 sequences with the help of Professor Harmer from the University of Exeter Living Systems Institute. The final alignment of the sequences was then fed into the ANCESCON software that performed the ancestral reconstruction. The software produced a phylogenetic tree and identified 74 ancestors. In order to identify the most suitable ancestors to model and use, we used the method below to weight each one:
+<br>
+Following this method, we identified 4 potentially suitable ancestors to further analyse. The YASARA software was used to model the 3D structure of each of the four ancestors. The models produced were then aligned against the structure of PETase by Professor Harmer in order to identify significant changes in the sequences. We discovered that the catalytic triad was conserved in all four ancestors, suggesting that the PET degrading activity had not been lost. Interestingly, we have also discovered that a beneficial mutations reported in past papers was already present in all four of the ancestors, namely R280A. The only significant trait lost during the reconstruction was the second disulfide bond that was present in PETase but not the ancestors. However, we reverted this by changing the two alanine residues in the ancestors with two cysteine residues. Additionally, we deleted the first five amino acids from the N-terminus that we suspected were composing the signal peptide. Once these minor adjustments had been completed, we sent the final sequences to be synthesised.
+</p>
 ===Characterisation===