Team:HK GTC/Notebook
Notebook
31/1/2019
Miniprep for two PETase constructs:
Result: Show desired band when cut
- Wild Type
- W159H/S238F
Result: Show desired band when cut
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
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
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
Grow overnight culture for PETase in PET21b and for positive control in PET28a
Colony PCR to confirm transformation success
Result: Positive
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:
Result of SDS-PAGE:
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
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.
RE Digestion with XbaI results:
RE Digestion with XhoI, Bgl II results:
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.
RE Digestion with XbaI results:
RE Digestion with XhoI, Bgl II results:
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
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:
SDS-PAGE gel of whole cell protein of constructs 1,3,4,5.
SDS-PAGE results:
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.
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
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.
Protein extraction and SDS-PAGE for large scale culture
Result: No obvious band in elution.
SDS-PAGE gel result: No obvious change of band thickness at desired kDa.
Protein extraction and SDS-PAGE for large scale culture
Result: No obvious band in elution.
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
SDS-PAGE gel of small scale with varying IPTG concentration
Small scale results: No obvious change in band thickness at different IPTG concentrations
Purified protein SDS-PAGE results: Thick band at desired kDa, indicates successful large scale induction and purification
SDS-PAGE gel of small scale with varying IPTG concentration
Small scale results: No obvious change in band thickness at different IPTG concentrations
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.
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
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
28/8/2019
Inoculation for:
- Wild Type
- W159H/S245I
- W159H/S214H
- W159H/S238F
29/8/2019
Induction for Wild Type and W159H/S238F at room temperature
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
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
Transformation for GFP (from kit plate) and PET28a
Result: No colonies
Result: Thick band for desired protein
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
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
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
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:
Enzyme assay mix (200 µl final volume):
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
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
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:
Band purification for GFP construct, and quantification of DNA using OD600 and A260/280 measurements
Result:
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
Gel purification and quantification for above DNA for further experiments
20/9/2019
Enzyme assay (same protocol as 13/9)
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:
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
Second enzyme assay (protocol: same as 13/9)
Results: Inconclusive, little activity
- 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
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
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
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
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
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)-
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.
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.
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-
Gel result: Mostly positive
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
Gel result: Mostly positive
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
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
Standard curve:
Final PNP concentration:
Results:
W159H/S245I shows highest activity, followed by W159H/S238F, then S245I, Wild Type, W159H/S214H, S245I/R280L
Standard curve:
Final PNP concentration: