iGEM 2019 — Team HK_GTC

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# Team:HK GTC/Notebook

* [Home](https://2019.igem.org/Team%3AHK_GTC)
* Team

  [Team Members](https://2019.igem.org/Team%3AHK_GTC/Team)
  [Attributions](https://2019.igem.org/Team%3AHK_GTC/Attributions)
* Project

  [Description](https://2019.igem.org/Team%3AHK_GTC/Description)
  [Design](https://2019.igem.org/Team%3AHK_GTC/Design)
  [Notebook](https://2019.igem.org/Team%3AHK_GTC/Notebook)
  [Experiments and Results](https://2019.igem.org/Team%3AHK_GTC/Results)
  [Future Perspective](https://2019.igem.org/Team%3AHK_GTC/Future_Perspective)
* Parts

  [Parts Overview](https://2019.igem.org/Team%3AHK_GTC/Parts)
* [Safety](2019.igem.org/Team%3AHK_GTC/Safety)
* Human Practices

  [Integrated Human Practices](https://2019.igem.org/Team%3AHK_GTC/Human_Practices)
  [Collaborations](https://2019.igem.org/Team%3AHK_GTC/Collaborations)
  [Education & Engagement](https://2019.igem.org/Team%3AHK_GTC/Public_Engagement)
* [Judging Form](https://igem.org/2019_Judging_Form?team=HK_GTC)

* [Home](https://2019.igem.org/Team%3AHK_GTC)
* Team

  [Team Members](https://2019.igem.org/Team%3AHK_GTC/Team)
  [Attributions](https://2019.igem.org/Team%3AHK_GTC/Attributions)
* Project

  [Description](https://2019.igem.org/Team%3AHK_GTC/Description)
  [Design](https://2019.igem.org/Team%3AHK_GTC/Design)
  [Notebook](https://2019.igem.org/Team%3AHK_GTC/Notebook)
  [Experiments and Results](https://2019.igem.org/Team%3AHK_GTC/Results)
  [Future Perspective](https://2019.igem.org/Team%3AHK_GTC/Future_Perspective)
* Parts

  [Parts Overview](https://2019.igem.org/Team%3AHK_GTC/Parts)
* [Safety](2019.igem.org/Team%3AHK_GTC/Safety)
* Human Practices

  [Integrated Human Practices](https://2019.igem.org/Team%3AHK_GTC/Human_Practices)
  [Collaborations](https://2019.igem.org/Team%3AHK_GTC/Collaborations)
  [Education & Engagement](https://2019.igem.org/Team%3AHK_GTC/Public_Engagement)
* [Judging Form](https://igem.org/2019_Judging_Form?team=HK_GTC)

<!DOCTYPE html>

Notebook

# Notebook

## 31/1/2019

Miniprep for two PETase constructs:

* Wild Type
* W159H/S238F

RE Digestion and screening for above constructs

Result: Show desired band when cut

![](https://static.igem.org/mediawiki/2019/1/1e/T--HK_GTC--31-1-19.png)

## Early March

Recruitment of iGEM team members

## 11/3/2019

SDS-PAGE for Wild Type and W159H/S238F PETase
Result: No obvious band for PETase

## 31/1/2019

Miniprep for our designed constructs:

* W159H/S214H
* W159H/S245I
* S245I/R280L
* S245I

RE Digestion and Colony PCR for above constructs

## Late April

Brainstorming outreach to NGOs and other iGEM teams for human practices and collaboration

## Early May

Start reaching out to NGOs and other local iGEM teams

## 8/5/2019

Induction of small scale culture of Wild Type and W159H/S238F PETase, with 1mM of IPTG, induced for 3 hrs
Colony PCR screening for above constructs.

## 10/5/2019

SDS-PAGE gel of whole cell protein of the two constructs above.

## Mid-May

Confirmed collaboration with team Hong\_Kong\_HKUST

## Early June

Start brainstorming outreach to professors with experience about plastic pollution for human practices

## 5/6/2019

First meeting with team Hong\_Kong\_HKUST, discuss our projects and how we can help each other

## Mid-June

Confirmed interviews with Prof. Stanley Lau and Prof. Christelle Not

Brainstorming our activity for education and engagement

## 17/6/2019

Transformation of WT PETase sequence within PET21b vector into C41 cells

## 18/6/2019

Transformation of construct sequences within PET21b vector into DH5ɑ cells

Colony PCR to confirm transformation success

Result: Positive

![](https://static.igem.org/mediawiki/2019/6/6a/T--HK_GTC--18-6-19.png)

Grow overnight culture for PETase in PET21b and for positive control in PET28a

## 19/6/2019

Protein induction for PETase with varying IPTG concentration (0, 0.5, 1mM), lasting 4 hours/overnight, and
SDS-PAGE

Result of SDS-PAGE:

![](https://static.igem.org/mediawiki/2019/7/7b/T--HK_GTC--19-6-19.png)

## 20/6/2019

Protein induction for PETase and positive control in PET28a with varying IPTG concentration (0, 0.5, 1mM),
lasting 3 hours and overnight

## 21/6/2019

Preparation of samples from 20/6 for SDS-PAGE

## 26/6/2019

SDS-PAGE for above samples

## 27/6/2019

Minipreparation of plasmid for all PETase constructs:

* Wild Type
* S245I
* S245I/R280L
* W159H/S245I
* W159H/S214H
* W159H/S238F

First interview with Prof. Christelle Not, mainly about the situation of plastic pollution in Hong Kong and
what
we can do to raise awareness

## 11/7/2019

Transformation of constructs into C41/DE3 competent cells.

* Wild Type
* W159H/S214H
* W159H/S245I
* S245I
* W159H/S238F
* S245I/R280L

## 16/7/2019

All colonies grow successfully except W159H/S214H.

Colony PCR of cells with construct 1,3,4,5 and 6 is performed.

RE Digestion with XbaI of all constructs is performed.

RE Digestion with XhoI, Bgl II of all constructs is performed

Colony PCR results: All positive.

![](https://static.igem.org/mediawiki/2019/3/37/T--HK_GTC--16-7-19_1.png)
![](https://static.igem.org/mediawiki/2019/f/f6/T--HK_GTC--16-7-19_2.png)
![](https://static.igem.org/mediawiki/2019/9/9c/T--HK_GTC--16-7-19_3.png)
![](https://static.igem.org/mediawiki/2019/4/4b/T--HK_GTC--16-7-19_4.png)
![](https://static.igem.org/mediawiki/2019/a/ae/T--HK_GTC--16-7-19_5.png)

RE Digestion with XbaI results:

![](https://static.igem.org/mediawiki/2019/5/5e/T--HK_GTC--16-7-19_6.png)

RE Digestion with XhoI, Bgl II results:

![](https://static.igem.org/mediawiki/2019/1/1a/T--HK_GTC--16-7-19_7.png)

## 17/7/2019

For constructs 1,3,4,5 and 6, 8 subcultures are made per construct in a small scale, and they are inoculated
in
LC medium.

Induction of protein by adding 0.5mM IPTG, shaken overnight

## 18/7/2019

Transformation of W159H/S214H into C41/DE3 competent cells.

SDS-PAGE gel of whole cell protein of constructs 1,3,4,5.

SDS-PAGE results:

![](https://static.igem.org/mediawiki/2019/7/77/T--HK_GTC--18-7-19_1.png)

![](https://static.igem.org/mediawiki/2019/5/59/T--HK_GTC--18-7-19_2.png)

![](https://static.igem.org/mediawiki/2019/a/a1/T--HK_GTC--18-7-19_3.png)

![](https://static.igem.org/mediawiki/2019/3/3d/T--HK_GTC--18-7-19_4.png)

## 19/7/2019

Second meeting with team Hong\_Kong\_HKUST, discuss our difficulties with protein induction and about their
model

Colonies of construct 2 grow successfully.

Induction of protein with different IPTG concentration of first subculture of the wild type colonies in
small
scale.

## 20/7/2019

Induction of wild type subculture with varying IPTG concentration (0, 0.01, 0.05, 0.1, 0.5mM)

Induction of large scale culture with 0.01mM IPTG for 5 hours

## 21/7/2019

Preparation of samples from 20/7 for SDS-PAGE

## 23/7/2019

SDS-PAGE gel for protein from wild type subculture induced with varying IPTG concentrations

SDS-PAGE gel result: No obvious change of band thickness at desired kDa.

![](https://static.igem.org/mediawiki/2019/0/08/T--HK_GTC--23-7-19_1.png)

Protein extraction and SDS-PAGE for large scale culture

Result: No obvious band in elution.

![](https://static.igem.org/mediawiki/2019/f/f2/T--HK_GTC--23-7-19_2.png)

## 24/7/2019

Preparation of overnight culture of PETase

## 25/7/2019

Subculture of PETase is made in large scale and induced at room temperature with IPTG overnight.

## 26/7/2019

Large scale protein purification with nickel resin and columns.

## 27/7/2019

SDS-PAGE gel of purified protein product is performed.

Purified protein SDS-PAGE results: Thick band at desired kDa, indicates successful large scale induction and
purification

![](https://static.igem.org/mediawiki/2019/f/f7/T--HK_GTC--27-7-19_1.png)

![](https://static.igem.org/mediawiki/2019/a/a0/T--HK_GTC--27-7-19_2.png)

SDS-PAGE gel of small scale with varying IPTG concentration

Small scale results: No obvious change in band thickness at different IPTG concentrations

![](https://static.igem.org/mediawiki/2019/7/75/T--HK_GTC--27-7-19_3.png)

## 7/8/2019

Subculture of S245I, S245I/R280L, and W159H/S245I is made in large scale and induced at room temperature
with
IPTG.

Glycerol stocks of cells of all constructs are made.

## 8/8/2019

Protein purification for S245I, S245I/R280L, and W159H/S245I with nickel resin and columns.

Glycerol stocks of all purified protein are made.

SDS-PAGE gel of protein product is performed.

SDS-PAGE results: Protein of interest is obtained for S245I, S245I/R280L. Negative result for W159H/S245I

![](https://static.igem.org/mediawiki/2019/f/f2/T--HK_GTC--8-8-19_1.jpg)

![](https://static.igem.org/mediawiki/2019/5/5e/T--HK_GTC--8-8-19_2.jpg)

![](https://static.igem.org/mediawiki/2019/5/5d/T--HK_GTC--8-8-19_3.jpg)

## 28/8/2019

Inoculation for:

* Wild Type
* W159H/S245I
* W159H/S214H
* W159H/S238F

Result: Only 1 and 4 have successful growth

## 29/8/2019

Induction for Wild Type and W159H/S238F at room temperature

Induction for Wild Type and W159H/S238F at room temperature

## 30/8/2019

Protein purification for Wild Type and W159H/S238F using nickel column

Desalting protein for Wild Type and W159H/S238F

Final meeting with team Hong\_Kong\_HKUST, discuss the generation of data for their model and the progress of
our
projects

## 2/9/2019

SDS-PAGE for purified Wild Type and W159H/S238F

Result: Thick band for desired protein

![](https://static.igem.org/mediawiki/2019/5/51/T--HK_GTC--2-9-19_1.jpg)

![](https://static.igem.org/mediawiki/2019/1/1b/T--HK_GTC--2-9-19_2.jpg)
Transformation for GFP (from kit plate) and PET28a

Result: No colonies

## 4/9/2019

Inoculation for

* W159H/S245I
* W159H/S214H
* S245I
* S245I/R280L

## 5/9/2019

Induction for above constructs at room temperature overnight

Transformation for GFP and PET28a for use in characterisation

## 6/9/2019

Protein extraction and purification for above constructs

## 11/9/2019

Colony PCR Screening for GFP with PET28a

Preparation of culture for all GFP constructs used for characterisation (PET28a RBS, RBS34, J61124)

Result: Only construct with PET28a has growth

## 12/9/2019

Colony PCR for constructs transformed on 5/9/2019

Result: No band in desired region

## 13/9/2019

Determination of concentration for proteins using Bradford assay (with BSA and Bio-Rad Bradford Assay Dye
reagent), and preliminary enzyme assay

Result of protein concentration determination:

* Wild Type: 1.418 µg/µl
* W159H/S245I: 0.4764 µg/µl
* S245I/R280L: 1.825 µg/µl
* S245I: 1.408 µg/µl
* W159H/S214H: 0.5969 µg/µl
* W159H/S238F: 1.418 µg/µl

Enzyme assay mix (200 µl final volume):

* 1mM p-nitrophenyl dodecanoate (substrate)
* 45mM sodium hydrogenphosphate (Na2HPO4)
* 90mM NaCl
* 10% DMSO
* 10 µg enzyme

200 µl:

2µl 0.1M substrate, 18µl 0.5M Na2HPO4, 3.6µl 5M NaCl, 20µl 100% DMSO, 10µg enzyme (volume depends on
concentration), make up to 200µl with exchange buffer

## 17/9/2019

PCR of GFP with RBS34 using PrimeSTAR max protocol, comparison of using 55℃ and 60℃ in cycle

Result:

![](ihttps://static.igem.org/mediawiki/2019/1/12/T--HK_GTC--17-9-19.png)

Band purification for GFP construct, and quantification of DNA using OD600 and A260/280 measurements

## 18/9/2019

RE Digestion of GFP with PET28a vector and RBS34 insert using XbaI, XhoI

Gel purification and quantification for above DNA for further experiments

## 20/9/2019

Enzyme assay (same protocol as 13/9)
![](https://static.igem.org/mediawiki/2019/2/2f/T--HK_GTC--20-9-19.png)

## 23-29/9/2019

Generation of data using Hong\_Kong\_HKUST’s mathematical model of their toggle switch

## 30/9/2019

Ligation of parts for use in characterisation:

* PET28a, and GFP with RBS24 and J61124
* Samples (all with 1 µl buffer and 1 µl ligase, mad up)-

+ 3 µl PET28a, 0.6 GFP+RBS34 µl insert
+ 2 µl PET28a, 0.6 GFP+RBS34 µl insert
+ 3 µl PET28a, 1 GFP+J61124 µl insert
+ 3 µl PET28a, 1.5 GFP+J61124 µl insert
+ 3 µl PET28a, 1 µl GFP+RBS34 insert
+ 3 µl PET28a, 1.5 µl GFP+RBS34 insert

Ligation condition: Room temp. overnight (about 19 hr this time)

RE Digestion of GFP constructs and PET28a, followed by band purification and quantification.

SDS-PAGE of all protein constructs for confirmation

SDS-PAGE results: All positive

![](https://static.igem.org/mediawiki/2019/2/2a/T--HK_GTC--30-9-19_1.png)

![](https://static.igem.org/mediawiki/2019/c/c3/T--HK_GTC--30-9-19_2.png)

![](https://static.igem.org/mediawiki/2019/1/15/T--HK_GTC--30-9-19_3.png)

![](https://static.igem.org/mediawiki/2019/7/74/T--HK_GTC--30-9-19_4.png)

Second enzyme assay (protocol: same as 13/9)

Results: Inconclusive, little activity

## 2/10/2019

Colonies from 30/9 are observed.

Colony PCR Screening for all ligated constructs from 30/9 is performed

Results: Almost all colonies have no desired band

2 positive colonies for sample 1, 1 for sample 2

![](https://static.igem.org/mediawiki/2019/3/35/T--HK_GTC--2-10-19.jpg)

## 10/10/2019

Glycerol stocks of cells with GFP+RBS34 plasmids are made (those with positive result from 2/10 PCR screen)

Miniprep to extract plasmid with RBS34 and GFP gene (from colonies with positive result from 2/10 PCR
screen),
and quantification

PCR (PrimeSTAR Max) of GFP+J61124 is performed
Agarose gel electrophoresis is performed with PCR products.
Gel result: All positive

Band preparation of PCR product is performed.
RE digestion of PET28a, J61124 with GFP and plasmid with RBS34 and GFP is performed.

PCR (with PrimeSTAR protocol) of GFP gene obtained from miniprep is performed
Agarose gel electrophoresis is performed with PCR products and GFP from miniprep
Gel result: All positive

## 11/10/2019

Quantification of band purified GFP+J61124 and PET28a vectors from 10/10 and 18/9

Ligation of PET28a and GFP+J61124 (from band purified samples prepared on 10/10 and 18/9) is performed

Mixes (all with 1µl buffer and 1µl ligase, make up with H2O to 10µl)-

* 3µl of 18/9 PET28a, 0.3µl GFP+J61124 insert, ligate for 1 hour at room temp.
* 3µl of 10/10 PET28a, 0.4µl GFP+J61124 insert, ligate for 1 hour at room temp.
* 3µl of 10/10 PET28a, same as sample 2 except ligate overnight at room temp.
* 3µl of 10/10 PET28a, 0.7µl GFP+J61124 insert, ligate overnight at room temp.

Band preparation and quantification of PCR product from 10/10

Miniprep and quantification of PET28a and GFP+J61124 is performed.

RE digestion (overnight) of PET28a, J61124+GFP with XhoI and XbaI is performed.

Transformation of ligated plasmid (sample 1 and 2) is performed.

## 12/10/2019

Colonies from 11/10 transformation are observed.

Band preparation of 11/10 RE digestion products (PET28a, J61124+GFP) is performed.

Ligation of PET28a and J61124 with GFP is performed

Mixes (All have 1µl buffer and 1µl ligase, make up to 10µl with H2O-

* 4µl PET28a, 0.7µl J61124+GFP
* 3µl PET28a, 0.9µl J61124+GFP

Colony PCR Screening of transformed cells with PET28a, J61124 with GFP is performed.

Gel result: Mostly positive

![](https://static.igem.org/mediawiki/2019/5/5e/T--HK_GTC--12-10-19.jpg)

## 14/10/2019

Testing new protocol for enzyme assay:

* E1: 1mM substrate, 45mM Na2HPO4-HCl buffer, 10µg Wild Type PETase
* E2: 2mM substrate, 45mM Na2HPO4-HCl buffer, 10µg Wild Type PETase
* E3: Protocol from 13/9, 10µg Wild Type PETase
* E4: Negative control for E1 with no enzyme
* E5: Negative control for E2 with no enzyme
* E6: Negative control for E3 with no enzyme

Result: E1 has the highest percentage increase in OD
![](https://static.igem.org/mediawiki/2019/8/84/T--HK_GTC--14-10-19.png)

## 15/10/2019

Enzyme assay for all constructs using new protocol (E1 from 14/10)

Results:

W159H/S245I shows highest activity, followed by W159H/S238F, then S245I, Wild Type, W159H/S214H, S245I/R280L

![](https://static.igem.org/mediawiki/2019/6/65/T--HK_GTC--OD_increase.gif)

Standard curve:

![](https://static.igem.org/mediawiki/2019/f/f7/T--HK_GTC--15-10-19_2.png)

Final PNP concentration:

![](https://static.igem.org/mediawiki/2019/9/99/T--HK_GTC--Final_PNP.png)


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# Team:HK GTC/Experiments

* [Home](https://2019.igem.org/Team%3AHK_GTC)
* Team

  [Team Members](https://2019.igem.org/Team%3AHK_GTC/Team)
  [Attributions](https://2019.igem.org/Team%3AHK_GTC/Attributions)
* Project

  [Description](https://2019.igem.org/Team%3AHK_GTC/Description)
  [Design](https://2019.igem.org/Team%3AHK_GTC/Design)
  [Notebook](https://2019.igem.org/Team%3AHK_GTC/Notebook)
  [Experiments and Results](https://2019.igem.org/Team%3AHK_GTC/Results)
  [Future Perspective](https://2019.igem.org/Team%3AHK_GTC/Future_Perspective)
* Parts

  [Parts Overview](https://2019.igem.org/Team%3AHK_GTC/Parts)
* [Safety](2019.igem.org/Team%3AHK_GTC/Safety)
* Human Practices

  [Integrated Human Practices](https://2019.igem.org/Team%3AHK_GTC/Human_Practices)
  [Collaborations](https://2019.igem.org/Team%3AHK_GTC/Collaborations)
  [Education & Engagement](https://2019.igem.org/Team%3AHK_GTC/Public_Engagement)
* [Judging Form](https://igem.org/2019_Judging_Form?team=HK_GTC)

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:HK GTC/Results

* [Home](https://2019.igem.org/Team%3AHK_GTC)
* Team

  [Team Members](https://2019.igem.org/Team%3AHK_GTC/Team)
  [Attributions](https://2019.igem.org/Team%3AHK_GTC/Attributions)
* Project

  [Description](https://2019.igem.org/Team%3AHK_GTC/Description)
  [Design](https://2019.igem.org/Team%3AHK_GTC/Design)
  [Notebook](https://2019.igem.org/Team%3AHK_GTC/Notebook)
  [Experiments and Results](https://2019.igem.org/Team%3AHK_GTC/Results)
  [Future Perspective](https://2019.igem.org/Team%3AHK_GTC/Future_Perspective)
* Parts

  [Parts Overview](https://2019.igem.org/Team%3AHK_GTC/Parts)
* [Safety](2019.igem.org/Team%3AHK_GTC/Safety)
* Human Practices

  [Integrated Human Practices](https://2019.igem.org/Team%3AHK_GTC/Human_Practices)
  [Collaborations](https://2019.igem.org/Team%3AHK_GTC/Collaborations)
  [Education & Engagement](https://2019.igem.org/Team%3AHK_GTC/Public_Engagement)
* [Judging Form](https://igem.org/2019_Judging_Form?team=HK_GTC)

# Experiments and Results

In this project, we designed 4 mutants of the enzyme, PETase by altering their amino acid sequence. In
which one of them is a single mutant and the other three are double mutants, hoping to enhance their function
in PET degradation process.
These changes include increasing hydrophobicity property and make the substrate binding site, subunit II more cutinase-like. This design rationale comes from a review of all previous attempts at site-directed mutagenesis of PETase. A clear trend in most successful mutation attempts is that an increase in hydrophobicity or a binding
site similar to *T. fusca* cutinase, which is narrower.

### The choice of bacterial cells As PETase is toxic to most lines and strains of Escherichia coli if it is overexpressed, it causes the death of cells if it takes up a certain portion of whole cell protein. So, a specialised cell strain, C41(DE3) which is derived from BL21/DE3 is used to perform protein induction and expression to prevent cell death, maximising the yield of PETase produced. We used DH5ɑas host cells to prepare DNA of 6 constructs as it has a higher insert stability and a lower chance of degrading plasmid during the miniprep process. The extracted DNA were then verified and transformed into C41(DE3) strain. The choice of vector To insert our gene of interest into the cells, we decided to use pET-21b plasmid as a vector. In the plasmid, T7 promoter is present which allow high level of gene expression and therefore a higher yield of gene product, PETase. Also, the plasmid has a high copy number which helps in passing the plasmid to daughter cells when they perform cell division, ensuring that our gene is present in all of the population in colonies. The pET-21b vector also contains a lac operon in front of the PETase gene. This reduces basal expression of PETase in normal condition and prevent leaky expression as it is highly effective. And our PETase protein would only be expressed when IPTG is added to the culture medium, so it prevents the overexpression which may lead to cell death in uninduced condition. Restriction enzyme digestion and colony PCR screening To ensure that our plasmids contained the gene and they are successfully transformed into the C41(DE3) bacterial cells, we performed both RE digestion and colony PCR screening to confirm it, before starting the next stage of experiment. For RE digestion, as there are RE sites in front, in between and at the back of the gene sequence, which are sites for XhoI, XbaI and BglII respectively. Applying the restriction enzymes will cut these sites and produce DNA fragments of different size, which can then be separated through agarose gel electrophoresis, to ensure that our plasmid actually contained the gene. ![](https://static.igem.org/mediawiki/2019/e/e9/T--HK_GTC--11.jpg) Figure 1. Restriction enzyme digestion analysis of uncut and NdeI/XhoI cut WT and W159H/S238F DNA. ![](https://static.igem.org/mediawiki/2019/d/d8/T--HK_GTC--22.jpg) Figure 2. Restriction enzyme digestion analysis of PETase constructs with XbaI. As seen above, the presence of bands near desired size indicates the presence of desired gene in the plasmid. We often use RE digestion to confirm sequences, or after ligation. Primers that can amplify the PETase gene of 300 bp is used to perform colony PCR. This can help us to confirm that the bacterial cells contain our gene of interest. If the PETase gene is present, the primer would be able to bind to the gene during the annealing processes, and this would lead to a positive band result after performing agarose gel electrophoresis. A ![](https://static.igem.org/mediawiki/2019/1/11/T--HK_GTC--44.jpg)B![](https://static.igem.org/mediawiki/2019/c/cc/T--HK_GTC--33.jpg)C ![](https://static.igem.org/mediawiki/2019/9/9b/T--HK_GTC--55.jpg) D ![](https://static.igem.org/mediawiki/2019/8/87/T--HK_GTC--66.jpg) E ![](https://static.igem.org/mediawiki/2019/a/aa/T--HK_GTC--77.jpg) Figure 3. Colony PCR screening of PETase constructs. A. S245I/R280L; B. WT; C. S245I/R280L; D. W159H/S238F; E. W159H/S245I We have extensively used colony PCR in our experiments. It is used to confirm the presence of desired plasmids in cells or confirm ligation success. Protein Purification As PETase expressed stays inside the cell instead of being secreted out, in order to obtain the enzyme, we would need to obtain the cell lysate by using lysis buffer and sonicating the cells. In normal situation, our protein will be present in the supernatant after centrifugation but not in the resuspends where inclusion bodies caused by overexpression stays. Our PETase is fused with a C-terminal 6XHis-Tag which is outside of its globular structure where the other cellular proteins do not. Histidine is capable to form dative covalent bond with nickel(II) ion. Thus, we used nickel resin and columns to separate the enzyme from other cellular protein in the supernatant, as they won’t bind to the column and will not be captured. After large scale induction and purification of protein, SDS-PAGE gel is performed to separate the protein by mass, as the SDS used can unfold the protein back to its to a linear structure and add negative charges to the protein by mass. When an electric field is applied, the protein will move towards the anode due to electrostatic attraction. Protein molecules with different mass and size move at a different speed, and therefore are separated. PETase is around 30-34kDa, and it will show a very thick band if the protein is overexpressed. In the elution of columns, a sharp thick band is observed, and the size of band is near to the size of PETase, indicating the successful expression and purification of PETase. Protein induction As IPTG is added to induce protein expression, at first, we followed the protocol of a paper, adding 0.5mM of IPTG to 1 ml small scale cultures for induction. Then we perform SDS-PAGE using whole cell protein to see if PETase (which is about 30-34kDa) is successfully expressed. A ![](https://static.igem.org/mediawiki/2019/d/d9/T--HK_GTC--88.jpg)B![](https://static.igem.org/mediawiki/2019/2/2e/T--HK_GTC--99.png)C ![](https://static.igem.org/mediawiki/2019/a/a1/T--HK_GTC--1010.png) D ![](https://static.igem.org/mediawiki/2019/1/1c/T--HK_GTC--1011.png) Figure 4. SDS PAGE of PETase proteins induced in 1ml culture with 0.5 mM IPTG for 16 hours at 37oC. A. W159H/S238F; B. WT; C. S245I; D: W159H/S245I A ![](https://static.igem.org/mediawiki/2019/9/9b/T--HK_GTC--12.jpg)B![](https://static.igem.org/mediawiki/2019/c/cb/T--HK_GTC--13.jpg) Figure 5. SDS PAGE of WT PETase proteins. WT PETase was induced in 1ml culture with 0, 0.01, 0.05, 0.1, 0.5 and 1 mM IPTG for 16 hours at A. room temperature; B. 37oC. ![](https://static.igem.org/mediawiki/2019/c/cc/T--HK_GTC--15.jpg) Figure 6. SDS PAGE of WT PETase proteins induced in 1ml culture with 0, 0.5 and 1 mM IPTG for 4 hours and 16 hours at 37oC. ![](https://static.igem.org/mediawiki/2019/6/6e/T--HK_GTC--14.jpg) Figure 7. SDS PAGE of WT PETase proteins induced in 100 ml culture with 0.01 mM IPTG for 16 hours at 37oC. As seen above, if the protein is successfully expressed, a thick band of 30 kDa should be seen, just like the one in positive control. Yet there are no positive bands in the whole cell protein we obtained from cell lysate. To optimise the conditions for protein expression we performed both small scale induction of protein with different IPTG concentration (0 mM ,0.01mM ,0.05mM ,0.1mM ,0.5mM), different induction time (4 hours and 16 hours), different temperature (37oC and room temperature) and large scale induction with 0.05mM IPTG. Then we performed large scale protein expression with 0.5mM of IPTG at room temperature. The protein of all constructs except W159H/S245I and W159H/S214H were successfully expressed. For these two constructs, 0.05mM of IPTG is later found to be optimized for inducing the protein expression. In summary, after trials of changing different IPTG concentration, temperature, duration and scale of culture, we obtained the optimised protocol to perform the protein purification. The condition was 100 ml culture induced with 0.5mM and 0.05mM IPTG at room temperature for 16-20 hours. A ![](https://static.igem.org/mediawiki/2019/0/01/T--HK_GTC--16.jpg)B![](https://static.igem.org/mediawiki/2019/c/c7/T--HK_GTC--17.jpg)C ![](https://static.igem.org/mediawiki/2019/3/36/T--HK_GTC--18.jpg) D ![](https://static.igem.org/mediawiki/2019/7/7e/T--HK_GTC--19.jpg) E ![](https://static.igem.org/mediawiki/2019/1/1b/T--HK_GTC--20.jpg)F![](https://static.igem.org/mediawiki/2019/3/38/T--HK_GTC--21.jpg) Figure 8. SDS PAGE of PETase proteins induced in 100 ml culture with 0.5 mM (A-D) and 0.05 mM (E-F) IPTG for 16 hours at room temperature. A. WT; B. S245I; C. W159H/S238F; D. S245I/R280L; E. W159H/S245I; F. W159H/S214H. Comparing the flow through of column with cell lysate of induced bacteria which contains whole cell protein, we can observe that the flow through have a thinner band at the size similar to PETase, showing that our protein is trapped in the column and is eluted out in elution. As seen from above, all constructs show a correct size bands after elution. This indicates a successful expression and purification for all of them. The proteins were then desalted and stored at -80oC. Bradford protein assay To know the concentration of our enzyme produced, we performed Bradford protein assay, in which Bio-Rad Protein Assay Dye Reagent Concentrate is added to the enzyme. When the dye binded with the protein, its colour shifts from brown to blue. The unbound form of dye absorbs light at a wavelength of 465nm but the bounded form of dye absorbs those at 595nm. By using plate reader to measure the OD595, we are able to determine our protein concentration when comparing its OD reading with that of protein concentration standards generated with BSA protein. After knowing that, we can then use a fixed mass of enzyme in the further steps such as enzyme assay, to ensure a fair experiment. A ![](https://static.igem.org/mediawiki/2019/4/40/T--HK_GTC--23.jpg)B![](https://static.igem.org/mediawiki/2019/f/f9/T--HK_GTC--24.png) Figure 9. Protein concentration determination. A. BSA standard curve; B. OD reading and calculated concentration of purified proteins measured by iMark Microplate Reader S/N 16691. Enzyme assay After obtaining the purified enzyme, 4-nitrophenyl dodecanoate is used as substrate to test the enzyme activity, as PETase work by cleaving the ester bond between PET which 4-nitrophenyl dodecanoate also have. After the cleavage, the para-nitrophenol (p-NP) produced can emit light at a wavelength of 415nm after excite. When the enzyme activity is higher, more para-nitrophenol is produced, the more light is emitted and the optical density increases. By using the plate reader to monitor optical density over time, we are able to measure the relative enzyme activity. We didn’t use PET film in experiment at the moment as they would require the constant monitoring of crystallinity of the plastic structure or pits generated on the film which requires using LC-MS or SEM. As 4-nitrophenyl dodecanoate have a structure similar to PET and we can measure the amount of its product released in degradation process by using a plate reader for measuring OD. Using it as a substrate would be easier for us to perform enzyme assays. ![](https://static.igem.org/mediawiki/2019/0/0e/T--HK_GTC--25.jpg) Figure 10. Enzyme assay results using 10% DMSO in the reaction mixture. As seen above, the first enzyme assay was inconclusive, as there's only very slight fluctuation of the OD value in which we believed as background noise. So we decided to check on the protocol we followed. In that protocol, 10% of DMSO is added to dissolve the substrate, but DMSO can affect the enzyme conformation and thus its effect. Therefore, we tried different combination of substrate concentration, presence or absence of DMSO and NaCl in reaction mixtures to work out the optimised protocol. The different combinations of components in reaction mixtures are as follows: E1: 1mM substrate, 45mM Na2HPO4-HCl buffer, 10µg WT PETase E2: 2mM substrate, 45mM Na2HPO4-HCl buffer, 10µg WT PETase E3: 1mM substrate, 45mM Na2HPO4-HCl buffer, 10μg WT PETase, 10% DMSO, 90mM NaCl E4: Negative control for E1 with no enzyme E5: Negative control for E2 with no enzyme E6: Negative control for E3 with no enzyme ![](https://static.igem.org/mediawiki/2019/7/73/T--HK_GTC--26.jpg) Figure 11. Enzyme assay results using different combination of components in the reaction mixtures. As a result, E1 has the highest percentage increase of OD reading, and therefore we used this condition for subsequent enzyme assays. In summary, after trials of changing DMSO concentration and substrate concentration, we obtained the optimised protocol to perform the enzyme assay. The condition was using 1mM substrate, 45mM Na2HPO4-HCl buffer and 10µg WT PETase. Then, we used our 6 PETase constructs to perform the enzyme assay with the optimised protocol. In the graph below, the percentage increase of OD is plotted against time. Using the percentage increase can help us neglect the initial OD of each reaction well, which may vary slightly. In the first 15 minutes, we measured the OD once per minute. Then, we measured the OD at the 30, 45, 60, 90, 105,120,140 and 160 minutes. Using these data, we generated the graph which reveals our result. ![](https://static.igem.org/mediawiki/2019/1/11/T--HK_GTC--27.jpg) Figure 11. A graph showing OD415 reading for all PETase constructs in enzyme essay. ![](https://static.igem.org/mediawiki/2019/6/65/T--HK_GTC--OD_increase.gif) Figure 12. A graph showing % increase of OD415 in enzyme essay of PETase constructs. ![](https://static.igem.org/mediawiki/2019/f/f0/T--HK_GTC--29.jpg) Figure 13. A table showing final % increase in OD415 of different PETase. The results showed that mutant W159H/S245I has the highest enzyme activity followed by W159H/S238F and S245I. All of them exhibited higher activity than WT. Then we constructed a standard curve of final product using 0, 50, 100, 150 and 200 uM p-NP in order to find out the concentration of products degraded by different PETase. ![](https://static.igem.org/mediawiki/2019/8/84/T--HK_GTC--30.jpg) Figure 14. A standard curve of different concentration of p-NP. Then we use the standard curve to calculate the final concentration of p-NP: ![](https://static.igem.org/mediawiki/2019/9/99/T--HK_GTC--Final_PNP.png) Figure 15. Concentration of p-NP in reaction mixture of different PETase. These results showed that W159H/S245I exhibited highest concentration of p-NP followed by W159H/S245I and W159H/S238F which agree with the results for percentage increase in OD. As shown above, the mutant W159H/S245I have the highest percentage increase in OD over time, also showing that is has the highest enzymatic activity among all enzyme variants as more products are produced. The mutated amino acid residues located in the substrate binding site of subsite II which bound with MHET through hydrophobic interaction. The result could be caused by the double mutant makes the substrate binding site in subsite II more cutinase-like and increases the hydrophobic property of the enzyme. By referring to the induction condition, interestingly, the expression of this mutant requires exceptionally low concentration of IPTG for induction. This could be caused by the production of mutant with enhanced PETase performance can greatly disrupts the normal metabolic activity of the cells, leading to cell death. W159H/S238F was designed by previous researchers showed enhanced activity of PETase. Our results showed this mutant had higher activity on PET than that of wild type PETase which was consistent with their findings. S245I, the single mutant also showed an increase in the enzymatic activity compared to that of the wild type PETase.The mutated amino acid residues located in the substrate binding site of subsite II which bound with MHET through hydrophobic interaction. And the amino acid changed made the substrate binding site in subsite II more cutinase-like and increases the hydrophobic property of the enzyme. The mutant W159H/S214H shows a similar activity to that of the wild type PETase. The mutant S245I/R280L shows decreased activity compared to that of wild type PETase. Also, from literature reviews, we can see that W159H always have a synergistic effect with another amino residue changed when a double mutant is made. Such as W159H/S238F double mutant. There was single mutant of S238F made by site-directed mutagenesis in which its enzymatic activity is lower than that of the wild type PETasse. When it is mutated with W159H, we can see a significant increase in the double mutants’ activity compared to that of wild type PETase. So, W159H is likely to have a synergistic effect, increasing the enzyme activity when it is associated with another site mutation. And the W159H/S245I double mutant also supports the claim. In summary, we optimized protocols of protein induction and enzyme essay of WT and mutants of PETase. We successfully purified and quantified recombinant proteins for enzyme assays. Of the 4 mutants that we designed, 2 of them S245I and S245I/W159H exhibited higher enzyme activity than WT PETase. W159H may have a synergistic effect on S245I mutant so that S245I/W159H exhibited higher activity than S245I single mutants. Thus, protein engineering approach of rational modification of certain residues on PETase opens the possibility for efficient degradation of PET plastic waste in an industrial process.