Difference between revisions of "Team:Paris Bettencourt/Project/Enzyme"

Abstract

Motivation and Background

Nature, from inspiration to a canvas

Polyphenol degrading enzymes

Figure 1: Targets of our enzymes within the malvidin molecule This figure shows the place in which each of the enzymes attack the malvidin molecule. The different coloured lightbolts represent the different enzymes.

Fabric Binding Domain fusion proteins - enhancing of wine stain degrading properties

Results

Outline of the project

Figure 2: Outline of the enzyme project The enzyme sub-project has two main parts: the first one is the testing of a set of polyphenol degrading enzymes on their ability to degrade Anthocyanins, and the second one is the directing of that ability for stain removal purposes by fusing the enzymes to the Fabric Binding Domains (FBD) found by our team.
First, the different genes are codon optimised and cloned in E. coli. The heterologously expressed enzymes are then tested with their natural substrate to check whether they are functional of not.
Once their functionality is confirmed, we move on to the fusion of the enzymes with the FBD. To test whether it is feasible to fuse proteins with the FBD without disrupting their activity, we first create GFP-FBD fusion proteins for each of the FBDs and test their fluorescence. We also test the affinity of the resulting fusion proteins on different fabrics. This allows us to confirm whether it is feasible to construct fusion proteins in which both the enzyme and the FBD's activities are functional.
Having confirmed that fusion proteins are able to maintain both their enzymatic activity and their binding activity, we create fusion proteins with our polyphenol degrading enzymes and the FBDs. We test the activity of the resulting fusion proteins on the natural substrate of each of the original enzymes.
The last step is to test the original enzymes and the GFP-fusion versions on their ability to degrade Anthocyanins. We envision doing this in two ways: by testing them on Malvidin directly and by testing them on fabrics stained with wine.

Confirming expression of our proteins

After obtaining E. coli BL21(DE3) strains able to express our polyphenol degrading enzymes, we decided to focus our work on four of them, BpuI, CatA, XylE and BG1. We decided to postpone the experiments with PPO2, since due to its origin in plants, its expression in E. coli was the most demanding out of the set of proteins. We also decided to postpone the experiments on TanLpI due to the fact that it forms inclusion bodies, which ones again demands a complicated setup to make functional.
The first step was to check whether we were correctly expressing our proteins of choice. Figure 3 shows an SDS-PAGE analysis of the cell extracts performed with the cells in which expression of the proteins was induced. The cell extract protocol can be seen in the methods part of this project.

Figure 3: SDS-PAGE gels for expression of BpuI, CatA and XylE. Sample preparation: E.coli BL21(DE3) cells expressing the proteins were induced for 5 hours with 0.5mM IPTG. After5 hours, the OD(600nm) was measured, the cells centrifuged and the pellet was resuspended in Laemmili sample buffer to a final OD of 10. The cells were cooked at 95ºC for 10 min, and 10uL of the resulting solution were loaded on the gel. Ladder used: Kaleidoskope. Staining: The gel was washed 3x with miliQ water to remove the SDS and staining was performed using BioRad Comassie for 30min. De-staining was performed by leaving the gel in miliQ water for 1 hour with gentle shaking.
As expected, no overexpression was observed in BL21(DE3), whether it was induced or not induced. BpuI was correctly overexpressed, with the observed size being the expected, around 59kDa. CatA overexpression was very mild, but the correct sized band was observed, of around 34kDa. XylE was correctly overexpressed, with the correct band being observed, of around 36kDa.

Overexpression of three of the four enzymes was achieved. BG1, although very promising, was not correctly expressed, and therefore we continued our experiments with BpuI, Cat and XylE alone.

Testing our enzymes in their natural substrate

The next natural step was to test whether the overexpressed proteins were functional. We performed cell extracts of BpuI, CatA and XylE enzymes and measured their activity using the appropriate substrates.

Figure 4: Confirmation of enzymatic activity of heterologously expressed BpuI, CatA and XylE. Results obtained using the cell extracts prepared as explained on the methods section of this page. Control corresponds at BL21(DE3) background, using cell extracts of BL21(DE3) cells that do not express our proteins. In all cases, values measured correspond to reaction product.
A. BpuI activity was measured in Citrate Phosphate Buffer at pH 4, with 0.4mM of ABTS being used as substrate, as recommended in the literature. Measurements were taken after 90min, timepoint at which the substrate present in the reaction medium was finished.
B. CatA activity was measured in Sodium Phosphate 50mM at pH 7, with 30mM of Catechol as substrate, as recommended in the literature. Measurements were taken after 35 min, timepoint at which all the substrate had been consumed.
C. XylE activity was measured in Potassium Phosphate 100mM at pH 7.5, with 30mM of Catechol as substrate, as recommended in the literature. Measurements were taken after 12 min, timepoint after which all the substrate had been consumed.

In all cases, the proteins were demonstrated to be functional. All three cell extracts carrying them were able to degrade their natural substrates more than the control.

Testing the effect of fusing FBD with proteins

The following step was to construct our fusion proteins, fusing Fabric Binding Domains to our best performing enzymes.
First, we needed to understand whether it was feasible to build a functional FBD-enzyme fusion protein. We needed to understand if the joining together of the two parts would have a negative effect on the binding capacity or in the enzyme itself.
An easy way of testing the feasibility of these constructs is to test the FBDs with GFP first. We constructed fusion proteins with the several FBD on the N'-terminal, since it was the terminal the binding domains were recommended to be placed at.

Figure 6: Analysis of the fusion proteins FBD-GFP. COMMENT THE IMAGE and say how we measured them

Having checked that the creation of these fusion proteins was possible, we proceeded to create four fusion proteins. We fused FBD1 (binds to all fabrics) and FBD10 (binds to cellulose) to CatA and XylE.

Figure 6: Confirmation of enzymatic activity of the fusion proteins CatA-FBD1, CatA-FBD10, XylE-FBD1 and XylE-FBD10. Results obtained using the cell extracts prepared as explained on the methods section of this page. Control corresponds at BL21(DE3) background, using cell extracts of BL21(DE3) cells that do not express our proteins. In all cases, values measured correspond to reaction product.
A. CatA fusion proteins' activity was measured in Sodium Phosphate 50mM at pH 7, with 30mM of Catechol as substrate, as recommended in the literature. Measurements were taken after 35 min, timepoint at which all the substrate had been consumed by the native protein.
B. XylE fusion proteins' activity was measured in Potassium Phosphate 100mM at pH 7.5, with 30mM of Catechol as substrate, as recommended in the literature. Measurements were taken after 12 min, timepoint after which all the substrate had been consumed by the native protein.

Figure 6 can have two interpretations: either the binding of the FBDs affects the activity of the proteins, or it lowers their expression. Both interpretations would explained the decrease in the activity that we observed. Nonetheless, the enzymes were still active.

Testing our enzymes on their ability to degrade Anthocyanins

The next step is to test our enzymes fused with the FBDs on their ability to degrade anthocyanins. A similar assay to the one performed with Catechol will be performed using Malvidin or a solution of Anthocyanins.

Testing our enzymes on their ability to degrade wine stains

Following the assay with the Anthocyanins, a similar assay to that performed with the fabric assays on the FBD-CBD fusion proteins will be performed, only this time it will be carried out with fabrics stained with wine, on the assay developed by the assay team and analysed with the software designed by the same team as well.

Methods

Codon optimisation of plant and bacterial genes of interest

One important step when expressing heterologously genes in E. coli, specially those of eukaryotic origin, is to perform Codon Optimisation. Different organisms have different codon preferences, which means that each organism has different abundances of the several codons that translate to a same amino acid.
We performed Codon Optimisation using the IDT Codon Optimisation tool.

Golden Gate and Gibson Assembly for Biobrick Construction

We built our plasmids using either Golden Gate or Gibson Assembly.
In the case of Golden Gate, we used NEB’s Golden Gate Assembly mix. 40fmol of backbone and 40fmol of insert were mixed in a 10uL total reaction, following the instructions provided by NEB. The mixure was incubated with the following cycle: 37ºC for 5 min, (37ºC for 2 min, 16ºC for 5min)x50, 37ºC for 5min, 50ºC for 10min and 80ºC for 10min.
In the case of Gibson Assembly, 200ng of DNA (backbone and insert with a ratio of 1:2) were mixed in a 15uL total reaction, using NEB’s 2X Hifi DNA Assembly MasterMix. The mixure was incubated 1h at 37ºC.

Induced protein expression by the use of expression systems

We used E. coli BL21(DE3) as our expression strain. Cells transformed with our constructs were incubated at appropriate temperature until they reached an OD(600nm) of 0.4-0.6. They were then induced with 0.5mM IPTG for 5 hours.

Preparation of cell extract for protein activity testing

We performed our cell extract preparation using ThermoScientific’s B-PER Bacterial Protein Extraction Reagent with Enzymes.

His-tag purification of proteins

We performed the purification of our proteins using ThermoScientific’s His-Pur Ni-NTA Resin and all buffers as indicated by the manufacturer.

SDS-PAGE analysis

We analysed our proteins using pre-made, 10% SDS-PAGE gels from BioRad.

Assaying the binding strength of the Fabric Binding Domains.

We assayed the strength or the Fabric Binding Domains using the assy developed by our team, carrying cotton, wool, silk and linnen untreated, unbleached fabrics. Cell extracts of BL21(DE3) strains carrying GFP-FBD fusion proteins were performed and 1:10 dilutions were made. The diluted cell extracts were incubated in the 96-well-plates-based assay with the fabrics overnight. Several washes of the fabrics were performed using water, PBS, BSA 5%, Ethanol 70% and Catechol 0.03M. The several measurements were performed in a Infinite M200-Pro Tecan plate reader.

Assaying the enhanced stain removal activity of fusion proteins

We assayed the stain removal activity of our fusion proteins in our assay, with the different fabrics stained with wine. The different measurements wera carried out in the Infinite M200-Pro Tecan plate reader.

Attributions

This project was done by Alicia Calvo-Villamañán and Sebastián Sosa-Carrillo. BpuI data was obtained by Mislav Acman. A special thanks to our advisor Nadine Bongaerts for all the insight on Golden Gate and for the GFP sequences. A special thanks to our advisor Jake Wintermute for all the help with the figures and the pDUET system. Thank you to Afonso Bravo for the help with the his-tag purification. Thank you to IDT for the synthesis of our genes, and thanks to NEB for the enzymatic kits.

References

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