iGEM 2019 — Team UNSW_Australia

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

# Team:UNSW Australia/Notebook

Team: UNSW Australia

[xml version="1.0" encoding="utf-8"?](/Team%3AUNSW_Australia)

[Home](/Team%3AUNSW_Australia)

Project

[Description](/Team%3AUNSW_Australia/Description)
[Design](/Team%3AUNSW_Australia/Design)

Experiment

[Overview](/Team%3AUNSW_Australia/Experiments)
[Scaffold](/Team%3AUNSW_Australia/Scaffold)
[PAM AND TYCA](/Team%3AUNSW_Australia/Pathway1)
[LXYL-P1-2 AND DBAT](/Team%3AUNSW_Australia/Pathway2)
[Protocols](/Team%3AUNSW_Australia/Protocols)
[Notebook](/Team%3AUNSW_Australia/Notebook)
[Safety](/Team%3AUNSW_Australia/Safety)

Modelling

[Overview](/Team%3AUNSW_Australia/Model)
[Creating 3D Structure Files](/Team%3AUNSW_Australia/ModellingGuide)
[Molecular Dynamics Analysis](/Team%3AUNSW_Australia/Molecular_Dynamics)
[Enzyme Kinetics](/Team%3AUNSW_Australia/Enzyme_Kinetics)

Human Practices

[Overview](/Team%3AUNSW_Australia/Human_Practices)
[Cancer](/Team%3AUNSW_Australia/Cancer)
[Sustainability](/Team%3AUNSW_Australia/Sustainability)
[Commercialisation](/Team%3AUNSW_Australia/Commercialisation)
[Integrated](/Team%3AUNSW_Australia/Integrated)

People

[Team](/Team%3AUNSW_Australia/Team)
[Collaborations](/Team%3AUNSW_Australia/Collaborations)
[Attributions](/Team%3AUNSW_Australia/Attributions)

Awards

[Entrepreneurship](/Team%3AUNSW_Australia/Entrepreneurship)
[Education & Public Engagement](/Team%3AUNSW_Australia/Public_Engagement)
[Medals](/Team%3AUNSW_Australia/Medals)
[Judging Form](https://igem.org/2019_Judging_Form?id=3075)

unsw.igem@gmail.com
[/unswigemteam](https://www.facebook.com/unswigemteam/)
[@unsw\_igem](https://www.instagram.com/unsw_igem/)

# Notebook

## Overview

All of our lab work were documented using Benchling. We've separated our lab work into cloning work and proteins expression & purification. Click on the buttons on the right side to toggle the lab notes for the respective categories.

## Week 1

### 15 April - 21 April

#### Protein Expression & Purification

* Overnight culture of alpha and beta prefoldins with catcher.
* Expression of prefoldins by IPTG induction in T7 express cells.
* Bug buster and SDS-PAGE gel run to check for protein size.
* Gel showed beta prefoldin as a strong band at the correct size however alpha was too small and possibly did not contain the fused catcher protein.

## Week 2

### 22 April - 28 April

#### Protein Expression & Purification

* Overnight culture of alpha and beta prefoldins with catcher.
* Expression of prefoldins by IPTG induction in T7 express cells.
* Freezing of samples

## Week 8

### 3 June - 9 June

#### Cloning

* PC2 lab induction.
* Construct lab plan, compile protocols and reagent list.

#### Protein Expression & Purification

* Lab induction
* Lab plan written
* Ordered materials

## Week 9

### 10 June - 16 June

#### Cloning

* Design and order G-blocks for DBAT, Duke's DBAT, LXYL-P1-2, TycA and PAM enzymes from IDT.

#### Protein Expression & Purification

* Meeting with supervisor to discuss lab plan

## Week 10

### 17 June - 23 June

#### Cloning

* Preparation of Luria Broth Agar with Ampicillin plates.
* Small scale grow up of pET19b plasmid containing competent Dh5-alpha E.coli cells.
* pET19b plasmid extracted and purified using Promega miniprep kit, plasmid concentration and purity quantified by nanodrop.

#### Protein Expression & Purification

* Building induction/ OGTR Induction.

## Week 11

### 24 June - 30 June

#### DNA

* Calculate PCR thermocycling conditions and mastermix contents.

#### Protein Expression & Purification

* Bug-buster and SDS-PAGE of samples prepared in week 2 (α-PFD + Catcher before and after IPTG induction, β-PFD + Catcher before and after IPTG induction).
* SDS-PAGE gels showed expression of all proteins was achieved in the soluble fraction however there was a low expression of alpha prefoldin, and beta-prefoldin was found in the insoluble fraction.
* Following successful expression results, cells were lysed via sonication by Nga, spun down and separated into soluble and insoluble fractions of proteins.
* Soluble and insoluble protein was prepared and incubated onto Ni-NTA beads (soluble α+β, insoluble α, insoluble β) then loaded onto gravity columns for elution by buffers with different imidazole concentrations into fractions.
* Elution fractions were run on SDS-PAGE to show presence of more pure protein.
* Made buffers for Ni-NTA Histrap protein purification (binding and elution buffers) and insoluble fraction denaturing (triton 1X and guanidine denaturing buffer).
* Made PC2 labels for chemicals.

## Week 12

### 1 July - 7 July

#### Cloning

* Linearise pET19b backbone by PCR amplification (Phusion DNA polymerase). The concentration and purity quantified by nanodrop, then visualised by agarose gel electrophorepsis.
* Rerun PCR of pET19b backbone with optimised annealing temperature.
* Circular plasmid DNA removed by DpnI digest (NEB), purified using QIAGEN PCR clean up kit.
* Gibson assembly of G-blocks into pET19b backbone, concentration and purity of DNA quanitified by nanodrop.
* Transformation of assembled constructs into competent T7 Express E.coli (NEB), then plated onto selection plates with Ampicillin and grown overnight.

#### Protein Expression & Purification

* Prepare Bolt MES buffer and protein fixative buffer (25% isopropanol, 10% acetic acid) for PAGE gels
.* Prepared elution buffer concentration gradient solutions for His-tag protein purification.
* Starter culture of prefoldins (α-PFD, α-PFD + Catcher, β-PFD, β-PFD + Catcher) in 10ml Luria broth overnight with ampicillin.
* Addition of starter cultures into separate autoclaved LB broths (1 L) with ampicillin at 1 X concentration with incubation at 37°C, 200 rpm (for fast growing) until 0.6 OD is reached.
* Cultures were induced with IPTG and incubated at 20°C, 200 rpm overnight (for protein production).
* Bug buster of induced and non-induced (control) samples of alpha, alpha +C, beta, beta +C, followed by SDS-PAGE to show protein production in response to IPTG induction and presence of proteins in soluble fractions (apart from beta and beta plus catcher which appeared in the insoluble fraction).
* Spin down culture to form pellets and freeze in -40°C.
* Filter and degas solvents to be used for AKTA in week 13.
* Training for AKTA start (chromatography machine for protein purification) and sonicator (for cell lysis).

## WEEK 13

### 8 July - 14 July

#### Cloning

* Gibson assembly of TycA into pET19b, troubleshooting molar ratio, transformed into T7 Express E.coli and grown overnight.
* Small scale grow up of DBAT, Duke's DBAT, PAM, LYXL and TycA.
* Extraction and purification of plasmids by miniprep (QIAGEN), quantified by nanodrop.

#### Protein Expression & Purification

* Cell lysis by sonication of pellet samples prepared in week 12
* Purify alpha +C, beta +C and alpha via AKTA start machine
* SDS-PAGE of fractions which showed clean proteins for all samples without contaminants however lots of protein was found in flow though (did not originally bind well to column)
* Concentration and buffer exchange of alpha +C, beta + C and alpha however beta + C precipitated in the concentration tube (may need to use dialysis for buffer exchange instead)
* BCA assay on beta +C, alpha w/ and without catcher to determine protein concentrations
* Reload alpha +C and beta +C flow-through on AKTA to collect protein found in original flow-through
* SDS page found flow through of second AKTA run still contained a lot of his-tagged protein not bound to column.

## Week 14

### 15 July - 21 July

#### Cloning

* Plasmid DNA concentrated using Promega DNA clean up Kit.
* Small scale grow up, miniprep (QIAGEN) of *TycA, PAM, Duke's DBAT* and *LXYL* containing *E.coli* strains.
* Samples submitted for sequencing by Ramaciotti Centre for Genomics.

## Week 15

### 22 July - 28 July

#### Cloning

* Replated *DBAT, PAM, TycA* and *LXYL* strains.
* Ordered *S. cerevisiae* codon optimised LXYL-P1-2 gBlock from IDT.
* Compiled reagent list and protocols for transformation of LXYL-P1-2 into *S. cerevisiae*.
* Preparation of YEPD media and agar plates.
* Obtained *S. cerevisiae BY4741* strain, courtesy of Marc Wilkin's Laboratory.

#### Protein Expression & Purification

* Run alpha flow-through on AKTA to collect protein found in original flow-through.
* Lyse E.coli cells containing beta prefoldin (from week 12 pellet samples) via sonication, and incubate washed Ni-NTA resin with supernatant (soluble protein).
* Wash resin and elute beta prefoldin from gravity column.
* SDS-PAGE, found beta prefoldin protein in fractions with limited contamination from other proteins.
* Concentration of alpha +C, alpha, beta +C and beta samples and flow-throughs.
* BCA assay for all four prefoldins to determine protein concentrations.

## Week 16

### 29 July - 4 August

#### Cloning

* Colony PCR of *DBAT, LXYL, TycA, PAM* and *Duke's DBAT*, amplified sequences visualised by gel electrophoresis.
* Designed primers to add "TAA" stop codon by PCR-directed codon addition of Duke's DBAT, primers ordered from Sigma Aldrich.

## Week 18

### 12 August - 18 August

#### Cloning

* PCR-directed codon addition of *Duke's DBAT*, visualisation of PCR product by gel electrophoresis and quantified by nanodrop.
* PCR-directed codon addition repeated with changes to annealing temperature and extension time.
* Ligation of LXYL *S. cerevisiae* pD1204 construct by Golden gate assembly and transformed into T7 Express *E.coli*.
* Gibson Assembly of *Duke's DBAT, TycA,* and *LXYL* into pET19b, pET19b and LXYL+pD1204 constructs transformed into T7 Express *E. coli*.* * The following was performed on the transformants: Small scale grow up, plasmid DNA extracted (QIAGEN miniprep kit), submitted for sequencing (Ramaciotti Centre for Genomics), colony PCR, gel electrophoresis and glycerol stocks for long term storage.

#### Protein Expression & Purification

* Starter cultures of alpha and beta prefoldins with and without catcher.
* Large scale grow-up and IPTG induction.
* Bug buster and SDS-PAGE found all samples contained protein but their was slightly less beta prefoldin protein than others.
* Pellets were harvested and stored in the -40°C freezer.

## Week 19

### 19 August - 25 August

#### Cloning

* Sequence alignment of constructs, unsuccessful assembly.
* Ordered Duke's DBAT gBlock with stop codon from IDT.

## Week 20

### 26 August - 1 September

#### Cloning

* Gibson assembly of *Duke's DBAT* and pET19b, construct transformed into T7 Express *E. coli*.
* DBAT, PAM, LXYL(e) and TycA plasmids submitted for sequencing. Insert amplified by PCR and visualised by gel electrophoresis.
* Colony PCR of LXYL(s), visualised by gel electrophoresis.
* Small scale grow up, miniprep and glycerol stocks of *Duke's DBAT, LXYL(e), LXYL(s)* and *TycA*.
* Research on possible assays for characterisation ATF1.

#### Protein Expression & Purification

* Purification of alpha +C, beta +C and alpha using AKTA start machine.
* Concentration of alpha +C, alpha, beta +C and beta samples and flow-throughs.
* Starter cultures of beta, PAM and DBAT prepared.
* Large scale grow-up and IPTG induction.
* PAM and DBAT pellets were harvested and stored in -40°C freezer.

## Week 21

### 2 September - 8 September

#### Cloning

* Colony PCR of LXYL(e), Duke's DBAT and TycA under optimal annealing temperatures and extension times
* Golden gate assembly of LXYL-P1-2 into pD1204 (provided by Joshua Hamey) and transformation into T7 Express *E. coli*
* Grow up of U, T, L(e), L(y), miniprep and samples submission for sequencing (Ramaciotti Centre)
* Gibson assembly of TycA+pET19b with time, concentration and gibson master mix controls. Transformed into T7 Express E. coli
* LXYL(e) gibson assembly and transformation
* Grow up of TycA and Duke DBAT and miniprep
* Prepared LB Agar and Chloramphenicol plates
* Transformed linear pSB1C3 and RFP+pSB1C3 into competent HB101 E.coli and plated
* Restreaked pink colonies on new plates to test plate batches

#### Protein Expression & Purification

* BCA assay for all four prefoldins to determine protein concentrations.
* Bug buster of PAM and DBAT showed good beta expression. However, lots of PAM in the insoluble fraction and no DBAT expression could be seen.
* Native page was performed on prefoldins to demonstrate assembly of assemblase hexameric scaffold. Successful results of assembly were achieved.
* Gravity column purification of PAM and DBAT yielded no soluble protein thus purification was unsuccessful.
* Purification of beta via AKTA start machine.

## Week 22

### 9 September - 15 September

#### Cloning

* Sequence alignment.
* Submit TycA#3 and Duke#1 for sequencing (Ramaciotti).
* LXYL(e) gibson assembly and transformation.
* Resuspended and transformed ATF1 (alcohol acetyltransferase) into HB101 E.coli, plated and grown overnight.
* Remade LB Agar + CAM plates.
* Troubleshoot Agar plates with RFP and linear controls and transformed into T7 Express E. coli.
* Troubleshoot CAM plates with known CAM resistant strains.
* Resuspended Red Luciferase and EPIC Luciferase DNA and transformed into T7 Express E.coli.
* Grow up of ATF, Red Luciferase and EPIC Luciferase.
* Colony PCR and miniprep of ATF, Red Luciferase and EPIC Luciferase. Visualised by gel electrophoresis.

#### Protein Expression & Purification

* Expression conditions were tested (IPTG and temperature) for PAM and DBAT.
* Bug buster of the different PAM and DBAT cultures were still insoluble.
* Purification by AKTA for PAM and DBAT samples show no protein binding and elution from the column.
* The Ellman’s assay was tested and a standard curve as constructed.

## Week 23

### 16 September - 22 September

#### Cloning

* Gel of TycA, Duke’s DBAT, LXYL+pET19b and LXYL+pD1204.
* Restriction enzyme digest of TycA, Duke’s DBAT, LXYL+pET19b, DBAT, PAM. Visualised by gel electrophoresis.
* Colony PCR of ATF colonies, imaged by gel electrophoresis.
* DpnI digest and PCR clean up of ATF4, ATF28 and ATF29 PCR products.
* Gibson assembly of ATF4, ATF28, ATF29 colonies and transformation into T7 Express cells.
* Colony PCR of ATF28/29, visualised by gel electrophoresis.
* Concentrate PAM and DBAT fractions and run on gel.

#### Protein Expression & Purification

* A 1L expression of alpha +C and beta +C was performed.
* More expression conditions for PAM and DBAT were tested.
* Bug buster shows expression of insoluble PAM while DBAT cultures contain both soluble and insoluble protein.
* SDS-PAGE of cell free lysate and soluble fraction of cultures reconfirms findings.
* Purification of alpha+C and beta+C as well as one PAM and one DBAT sample on AKTA.
* Buffer exchange of alpha+C and beta+C via dialysis.
* Concentration of PAM and DBAT fractions were run on gel showing very light PAM and DBAT bands.

## Week 24

### 23 September - 29 September

#### cloning

* Colony PCR of mCerulean3, TycA and ATF1 #28.
* DpnI digest (NEB) and DNA clean up (QIAGEN) of mCerulean3 and ATF1 fragments.
* Gibson assembly of mCerulean3, TycA, LXYL, Duke's DBAT and ATF1 into pET19b and transformed into T7 Express E.coli.
* Colony PCR of mCerulean3, TycA, LXYL, Duke's DBAT and ATF1. DNA fragments visualised by gel electrophoresis on a 1% agarose gel.
* Small scale grow up and miniprep of positive colonies.

#### Protein Expression & Purification

* Purified and non-purified PAM and DBAT bands were excised from SDS-PAGE gel and sent for mass spectrometry analysis. Results show both PAM and DBAT are present in the soluble fractions however PAM is at a very low concnetration.
* Expression of 1 L PAM cultures (0mM IPTG and 0.1 mM IPTG).
* Bug buster performed on PAM cultures. Both showed PAM in insoluble fraction.
* PAM (0.1 mM IPTG) was purified again on akta and fractions were concentrated and run on SDS PAGE gel
* Conjugation between PAM-Snooptag and beta prefoldin- SnoopCatcher was tested yielding negative results.
* Conjugation between alpha PFD and beta PFD on Native PAGE gel shows good results but need adjustments of protein ratios.

## Week 25

### 30 September - 6 October

#### Cloning

* Submitted Duke's DBAT, mCerulean3, ATF1 and TycA for sequencing (Ramaciotti Centre).
* Restriction enzyme digest of plasmid DNA. Visualised by gel electrophoresis.
* Sequence alignment check.
* Colony PCR of TycA. Amplicon visualised by gel electrophoresis.
* Preparation of 1000X Ampicillin stocks.
* Small scale grow up of PAM, TycA and gamma-prefoldin DNA. Plasmid DNA extracted and purified by miniprep (QIAGEN). DNA concentration and purity quanitified by nanodrop.
* Transformation of PAM into Origami 2(DE3)pLysS Competent Cells.

#### Protein Expression & Purification

* Purification of ATF1 for biobrick characterisation was performed.
* Assays on ATF1 activity were unsucessful.
* TycA, DUKE, ATF1 and mCerulean colonies were grown up, expressed and purified with DUKE, ATF1 and mCerulean showing expressed protein determined by bug buster.
* Grow up of alpha PFD beta PFD, PAM, DBAT and ATF1 was performed to keep pellets for later use.
* Beta and alpha PFD was purified on AKTA start machine.
* BCA assay on alpha PFD, beta PFD, mCerulean3 and mVenus gave approximate concentrations of proteins.
* Grow up of alpha PFD beta PFD, PAM, DBAT and ATF1 was performed to keep pellets for later use.
* Native Page was performed on alpha and beta PFD, yielding good results.
* Conjugation between alpha PFD and venus and beta PFD and cerulean yielded good conjugation.
* Concentration and buffer exchange of +c, b+b, mCerulean and Venus.
* Grew up alpha and beta and purified beta.
.* BCA assay was performed on a+c, b+b, mCerulean and Venus to determine protein concentration.
* Ellman assay of ATF1 was performed however was unsucessful
* SDS-PAGE gel of mCerulean3-SnoopTag with BPFD-SnoopCatcher and mCerulean3 (no tag) with BPFD-SnoopCatcher was run to show catcher-tag conjugation however one sample was incomplete.

## Week 26

### 7 October - 13 October

#### Protein Expression & Purification

* FRET was attempted twice however was unsuccessful.
* Native Page was performed on new alpha and beta PFD protein stocks yielding good results.
* Ellman assay of DBAT was performed however was unsucessful.
* Large scale grow up, espression and purification of PAM was performed.
* SDS-PAGE gel of mCerulean3-SnoopTag with BPFD-SnoopCatcher and mCerulean3 (no tag) with BPFD-SnoopCatcher was run to show catcher-tag conjugation yielding good results.
* Bug buster of DUKE, DBAT and PAM was performed.
* ATF1 was expressed, purified, concentrated, buffer exchnaged and run on BCA assay to determine protein concentration.
* Grow up and expression of PAM in Origami strain was performed twice however appeared to yeild no protein determined by bug buster.
* NMR spectra of alpha and beta-phenylalanine were taken as proof of concept for using NMR for PAM enzymatic assay.

## Week 27

### 14 October - 20 October

#### Protein Expression & Purification

* Expresseion and purification of DBAT using 6M urea on gravity column.
* Conjugation of DBAT-SnoopTag with BPFD-SnoopCatcher and PAM-SpyTag with aPFD-SpyCatcher using different concentration of Urea was unsucessful.
* SDS-PAGE gel of mCerulean3-SnoopTag with BPFD-SnoopCatcher and mCerulean3 (no tag) with BPFD-SnoopCatcher was run to show catcher-tag conjugation.
* Expression, purification, buffer exchange and concentration of ATF1 was performed.
* Expression and purification, of DBAT was performed.
* NMR spectra of deacetyl-paclitaxel was taken as proof of concept for using NMR for DBAT enzymatic assay.

Show All
Cloning Work
Protein Expression
Back to top

unsw.igem@gmail.com
[/unswigemteam](https://www.facebook.com/unswigemteam/)
[@unsw\_igem](https://www.instagram.com/unsw_igem/)


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

# Team:UNSW Australia/Experiments

Team: UNSW Australia

[xml version="1.0" encoding="utf-8"?](/Team%3AUNSW_Australia)

[Home](/Team%3AUNSW_Australia)

Project

[Description](/Team%3AUNSW_Australia/Description)
[Design](/Team%3AUNSW_Australia/Design)

Experiment

[Overview](/Team%3AUNSW_Australia/Experiments)
[Scaffold](/Team%3AUNSW_Australia/Scaffold)
[PAM AND TYCA](/Team%3AUNSW_Australia/Pathway1)
[LXYL-P1-2 AND DBAT](/Team%3AUNSW_Australia/Pathway2)
[Protocols](/Team%3AUNSW_Australia/Protocols)
[Notebook](/Team%3AUNSW_Australia/Notebook)
[Safety](/Team%3AUNSW_Australia/Safety)

Modelling

[Overview](/Team%3AUNSW_Australia/Model)
[Creating 3D Structure Files](/Team%3AUNSW_Australia/ModellingGuide)
[Molecular Dynamics Analysis](/Team%3AUNSW_Australia/Molecular_Dynamics)
[Enzyme Kinetics](/Team%3AUNSW_Australia/Enzyme_Kinetics)

Human Practices

[Overview](/Team%3AUNSW_Australia/Human_Practices)
[Cancer](/Team%3AUNSW_Australia/Cancer)
[Sustainability](/Team%3AUNSW_Australia/Sustainability)
[Commercialisation](/Team%3AUNSW_Australia/Commercialisation)
[Integrated](/Team%3AUNSW_Australia/Integrated)

People

[Team](/Team%3AUNSW_Australia/Team)
[Collaborations](/Team%3AUNSW_Australia/Collaborations)
[Attributions](/Team%3AUNSW_Australia/Attributions)

Awards

[Entrepreneurship](/Team%3AUNSW_Australia/Entrepreneurship)
[Education & Public Engagement](/Team%3AUNSW_Australia/Public_Engagement)
[Medals](/Team%3AUNSW_Australia/Medals)
[Judging Form](https://igem.org/2019_Judging_Form?id=3075)

unsw.igem@gmail.com
[/unswigemteam](https://www.facebook.com/unswigemteam/)
[@unsw\_igem](https://www.instagram.com/unsw_igem/)

# Experiments

scaffoldnotingrey11

## Click on the parts

### to see what we achieved

#### Scaffold

[![](https://static.igem.org/mediawiki/2019/d/df/T--UNSW_Australia--apfdlegend.png)

##### Alpha Prefoldin with SpyCatcher](/Team%3AUNSW_Australia/Scaffold)

[![](https://static.igem.org/mediawiki/2019/thumb/f/f6/T--UNSW_Australia--bpfdlegend.png/1198px-T--UNSW_Australia--bpfdlegend.png)

##### Beta Prefoldin with SnoopCatcher](/Team%3AUNSW_Australia/Scaffold)

#### Enzymes

[![](https://static.igem.org/mediawiki/2019/0/0c/T--UNSW_Australia--tycA-legend.png)

##### TycA with SpyTag

![](https://static.igem.org/mediawiki/2019/c/c6/T--UNSW_Australia--PAM-legend.png)

##### PAM with SnoopTag](/Team%3AUNSW_Australia/Pathway1)

[![](https://static.igem.org/mediawiki/2019/0/09/T--UNSW_Australia--lyxl-legend.png)

##### LXYL-P1-2 with SpyTag

![](https://static.igem.org/mediawiki/2019/5/5d/T--UNSW_Australia--dbat-legend.png)

##### DBAT with SnoopTag](/Team%3AUNSW_Australia/Pathway2)

map1

## Laboratory Objective #1 - Produce the Assemblase Scaffold

This is the first of our three laboratory objectives. We aim to express and purify βPFD-SnoopCatcher and αPFD-SpyCatcher. The assembly of these proteins into the Assemblase complex will then be demonstrated. Conjugation of SpyTag to SpyCatcher and SnoopTag to SnoopCatcher will also be demonstrated. To learn more see our [Assemblase Scaffold](https://2019.igem.org/Team%3AUNSW_Australia/Scaffold) page.

## Laboratory Objective #2 - Produce and test PAM and TycA of Pathway 1

Our second lab objective was to assemble and test pathway 1. We aimed to express and purify the reaction enzymes involved in the biosynthesis of the Paclitaxel side-chain; PAM-SnoopTag and TycA-SpyTag. These enzymes will then be conjugated to βPFD-SnoopCatcher and αPFD-SpyCatcher, respectively. The catalytic activity of the enzymes will also be tested. To learn more see our [Pathway 1](https://2019.igem.org/Team%3AUNSW_Australia/Pathway1) page.

## Laboratory Objective #3 - Produce and test LXYL and DBAT of Pathway 2

This is the third of our three laboratory objectives. We aim to express and purify the reaction enzymes involved in the alternative biosynthesis of Paclitaxel; DBAT-SnoopTag and LXYL-SpyTag. These enzymes will then be conjugated to βPFD-SnoopCatcher and αPFD-SpyCatcher, respectively. The catalytic activity of the enzymes will also be tested. To learn more see our [Pathway 2](https://2019.igem.org/Team%3AUNSW_Australia/Pathway2) page.

.

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

# Team:UNSW Australia/Results

Team: UNSW Australia

[xml version="1.0" encoding="utf-8"?](/Team%3AUNSW_Australia)

[Home](/Team%3AUNSW_Australia)

Project

[Description](/Team%3AUNSW_Australia/Description)
[Design](/Team%3AUNSW_Australia/Design)

Experiment

[Overview](/Team%3AUNSW_Australia/Experiments)
[Scaffold](/Team%3AUNSW_Australia/Scaffold)
[PAM AND TYCA](/Team%3AUNSW_Australia/Pathway1)
[LXYL-P1-2 AND DBAT](/Team%3AUNSW_Australia/Pathway2)
[Protocols](/Team%3AUNSW_Australia/Protocols)
[Notebook](/Team%3AUNSW_Australia/Notebook)
[Safety](/Team%3AUNSW_Australia/Safety)

Modelling

[Overview](/Team%3AUNSW_Australia/Model)
[Creating 3D Structure Files](/Team%3AUNSW_Australia/ModellingGuide)
[Molecular Dynamics Analysis](/Team%3AUNSW_Australia/Molecular_Dynamics)
[Enzyme Kinetics](/Team%3AUNSW_Australia/Enzyme_Kinetics)

Human Practices

[Overview](/Team%3AUNSW_Australia/Human_Practices)
[Cancer](/Team%3AUNSW_Australia/Cancer)
[Sustainability](/Team%3AUNSW_Australia/Sustainability)
[Commercialisation](/Team%3AUNSW_Australia/Commercialisation)
[Integrated](/Team%3AUNSW_Australia/Integrated)

People

[Team](/Team%3AUNSW_Australia/Team)
[Collaborations](/Team%3AUNSW_Australia/Collaborations)
[Attributions](/Team%3AUNSW_Australia/Attributions)

Awards

[Entrepreneurship](/Team%3AUNSW_Australia/Entrepreneurship)
[Education & Public Engagement](/Team%3AUNSW_Australia/Public_Engagement)
[Medals](/Team%3AUNSW_Australia/Medals)
[Judging Form](https://igem.org/2019_Judging_Form?id=3075)

unsw.igem@gmail.com
[/unswigemteam](https://www.facebook.com/unswigemteam/)
[@unsw\_igem](https://www.instagram.com/unsw_igem/)

scaffoldnotingrey11

## Legend

#### Scaffold

![](https://static.igem.org/mediawiki/2019/d/df/T--UNSW_Australia--apfdlegend.png)

### Alpha Prefoldin with SpyCatcher

![](https://static.igem.org/mediawiki/2019/thumb/f/f6/T--UNSW_Australia--bpfdlegend.png/1198px-T--UNSW_Australia--bpfdlegend.png)

### Beta Prefoldin with SnoopCatcher

#### Enzymes

![](https://static.igem.org/mediawiki/2019/0/09/T--UNSW_Australia--lyxl-legend.png)

### LXYL-P1-2 with SpyTag

![](https://static.igem.org/mediawiki/2019/5/5d/T--UNSW_Australia--dbat-legend.png)

### DBAT with SnoopTag

![](https://static.igem.org/mediawiki/2019/0/0c/T--UNSW_Australia--tycA-legend.png)

### TycA with SpyTag

![](https://static.igem.org/mediawiki/2019/c/c6/T--UNSW_Australia--PAM-legend.png)

### PAM with SnoopTag

## Introduction

The Assemblase scaffold is a fusion protein between the archaeal molecular chaperonin with SpyCatcher and Snoopcatcher, which allows for the attachment of 2 enzymes next to each other in a pathway especially if they control the rate-limiting step.

# Results

## Protein expression assay

Cells containing a plasmid with the αPFD-SpyCatcher or βPFD-SnoopCatcher insert (as confirmed by colony PCR and sequencing) were grown up in a large-scale overnight culture containing 1mM IPTG. A sample of this was used to perform a protein expression assay. Bug buster (a cell-lysis detergent) was then used to separate soluble and insoluble proteins.

![](https://static.igem.org/mediawiki/2019/c/cd/T--UNSW_Australia--1-PFD-bug-buster.jpeg )

**Figure 1.** Protein expression assay using bug buster to determine expression of alpha and beta prefoldin in soluble and insoluble forms. A: αPFD-SpyCatcher B: βPFD-SnoopCatcher

## Purification

Following the confirmation of protein expression by bug buster gels in Figure 1, αPFD-SpyCatcher and βPFD-SnoopCatcher were purified using the AKTA start machine via a Ni-NTA column.

![](https://static.igem.org/mediawiki/2019/3/3e/T--UNSW_Australia--2-PFD-PURIFICATION.jpeg )

**Figure 2.** SDS-PAGE of AKTA purification fractions (F3-9 and F11). A: αPFD-SpyCatcher B: βPFD-SnoopCatcher

## Assembly

Assembly of the hexameric Assemblase scaffold was visualized using a Native-PAGE gel as shown in Figure 3 below. The Assemblase scaffold (2 αPFD-SpyCatcher/4 βPFD-SnoopTag) can be seen to increase in concentration as more βPFD-SnoopCatcher is added to the αPFD-SpyCatcher, while αPFD-SpyCatcher decreases in concentration as increasing amounts of βPFD-SnoopCatcher is added.

![](https://static.igem.org/mediawiki/2019/thumb/8/8d/T--UNSW_Australia--3-Native-PAGE.jpeg/800px-T--UNSW_Australia--3-Native-PAGE.jpeg )

**Figure 3.** Native Page gel showing assembly of alpha and beta subunits into the hexameric Assemblase structure

## Conjugation

To show the function of the Spy and SnoopTag/catcher system, the fluorescent proteins, mVenus-SpyTag and mCerulean3- SnoopTag, were conjugated onto αPFD-SpyCatcher and βPFD-SnoopCatcher, respectively.

![](https://static.igem.org/mediawiki/2019/thumb/e/e7/T--UNSW_Australia--4-conjugation.jpeg/800px-T--UNSW_Australia--4-conjugation.jpeg )

**Figure 4.** SDS-PAGE showing conjugation of fluorescent protein to prefoldin (PFD). A: αPFD-SpyCatcher conjugated with mVenus-SpyTag. B: βPFD-SnoopCatcher conjugated with mCerulean-SnoopTag.

## FRET

We decided to use Förster resonance energy transfer (FRET) to demonstrate successful scaffolding of two different proteins to the α/β-PFD scaffold, as well as the self-assembly of the scaffold with proteins attached. Purified mCerulean3-SnoopTag and mVenus-SpyTag was conjugated to βPFD-SnoopCatcher and αPFD-SpyCatcher (respectively) in one tube to form the complex for FRET. Unfortunately, however, no FRET signal was detected as shown in Figure 5 below.

![](https://static.igem.org/mediawiki/2019/c/cd/T--UNSW_Australia--5-FRET-graph.png )

**Figure 5.** Emission spectrum from the FRET experiment run on assembled scaffold conjugated to fluorescent proteins and the fluorescent proteins by themselves

Initial assembly of the prefoldin scaffold before the addition of fluorescent proteins, as well as the conjugation of fluorescent proteins to their respective prefoldin prior to scaffold assembly were tested, however neither method showed any FRET. SDS-PAGE was run on the samples to visualize the conjugation of fluorescent proteins to the prefoldin scaffold.

![](https://static.igem.org/mediawiki/2019/thumb/e/e0/T--UNSW_Australia--6-FRET-gels.jpeg/799px-T--UNSW_Australia--6-FRET-gels.jpeg )

**Figure 6.** SDS-PAGE gel of samples tested using FRET analysis. A: Assembly of the scaffold performed prior to conjugation of fluorescent proteins. B: Tag/Catcher conjugation of proteins performed prior to scaffold assembly

## Discussion

A protein expression assay was used prior to purification in order to determine protein solubility. The bug buster gel in Figure 1A shows αPFD-SpyCatcher as soluble while Figure 1B shows βPFD-SnoopCatcher in the insoluble fraction. However, during purification, βPFD-SnoopCatcher appeared soluble. It is likely to have appeared insoluble in the bug buster protein expression assay due to the pH not being at pH8.

Following large scale grow up, the cells were induced with 1mM IPTG, pelleted and lysed by sonication, and the soluble fraction was purified on a Nickel NTA column via the AKTA start machine. Elution fractions thought to contain the protein (determined by observing the AKTA start chromatogram) were run on SDS-PAGE gel as in Figure 2. A large amount of contamination from unwanted proteins were seen in the early eluted fractions, however fractions 9-11 for αPFD-SpyCatcher and fraction 11 for βPFD-SnoopCatcher had little contaminants and were thus concentrated and buffer exchanged using an Amicon Ultra 15ml, 10KD concentrating column. Afterwards, protein concentration was determined using a BCA assay. This protein stock could then be used for further experiments as described below.

In order to show the formation of Assemblase, βPFD-SnoopCatcher was added to αPFD-SpyCatcher at an increasing concentration as the structure follows a ratio of 4 β prefoldins to two α prefoldins. These samples were run on a native page gel (shown in Figure 3) to allow the formation of the double β-barrelled bond formed between α and β prefoldin subunite. Assemblase is seen to increase in concentration as more βPFD-SnoopCatcher is added. This demonstrates the ability of our scaffold to self-assemble.

To establish the ability of our scaffold to conjugate proteins via the Spy and Snoop catcher/Tag system, the fluorescent proteins: mVenus-SpyTag and mCerulean-SnoopTag were expressed and purified. These were then added to αPFD-SpyCatcher and βPFD-SnoopCatcher respectively in different ratios and run on SDS-PAGE gel in Figure 4. Good conjugation is seen with s dark band observed at approximately 60kD (the molecular weight of prefoldin and fluorescent protein combined).

These recombinantly produced fluorescent proteins form a FRET pair, mCerulean3 as the FRET donor and mVenus as the FRET acceptor. These fluorescent proteins were conjugated to their respective prefoldins and combined to form the full hexameric structure. FRET was performed on the assembled structure as well as on appropriate controls, however no FRET was achieved as indicated in Figure 5. If the two proteins are brought close together via the scaffold and mCerulean3 is excited by a wavelength of 433 nm, its fluorescence emission (which roughly ranges from 475 to 515 nm) should excite mVenus and result in an additional fluorescence emission at 530 nm (mVenus emission), as well as a decrease in fluorescence at 475 nm (mCerulean3 quenching). This result was not achieved with no peak corresponding to mVenus emission being present in Figure 5.

Two strategies for scaffold conjugation and assembly were tested including assembling the scaffold prior to the simultaneous conjugation of mCerulean3 and mVenus via the Spy/Snoop catcher-tag system. The conjugation of αPFD-SpyCatcher and βPFD-SnoopCatcher to mVenus and mCerulean3 respectively prior to scaffold assembly was also tested with data shown in Figure 5. Both methods yielded similar results, with no increase in emission at 530nm relative to the emission at 475nm being observed.

Following this result, the samples were run on an SDS-PAGE gel (shown in Figure 6). Although the SDS-PAGE cannot show the full scaffold of Assemblase as all non-covalent bonds are denatured, any conjugation between fluorescent protein and prefoldin can be visualized. Figure 6 shows limited conjugation indicating a significant amount of free fluorescent proteins are present, which is likely to be the reason for no FRET signal. This result is unexpected as assembly and conjugation gels in Figures 3 and 4 indicate successful assembly between βPFD-SnoopCatcher and αPFD-SpyCatcher, as well as successful conjugation between fluorescent protein and prefoldin. Low conjugation observed in these FRET experiments may have occurred due to inadequate storage conditions of the fluorescent protein and prefoldin stock solutions. Due to time constraints, further tests could not be performed. However, in an effort to demonstrate complete assembly by FRET, future tests would include re-expression and purification of each protein. The quality of the assembly would then be verified on Native-PAGE and additional conjugation experiments would be performed in order to ensure appropriate ratios of scaffold to fluorescent proteins. Storage conditions should always be monitored with the proteins kept at 4 °C and these experiments should be performed within as little time as possible to avoid inactivation or precipitation of proteins, which may explain the inefficient Tag/Catcher conjugation observed towards the end of our project. Moreover, the complete assembly could be separated from excess prefoldin scaffold and/or free fluorescent proteins using size exclusion chromatography or multimodal chromatography. The pure hexameric, functionalised scaffold could then be used to perform FRET experiments and we would likely have more success due to no excess free fluorescent proteins (incapable of the FRET effect) being present in the reaction.

Title Here

abc

[Protein expression assay](#pcontent1)
[Purification](#pcontent2)
[Assembly](#pcontent3)
[Conjugation](#pcontent4)
[FRET](#pcontent5)
[Discussion](#pcontent6)
Back to top

unsw.igem@gmail.com
[/unswigemteam](https://www.facebook.com/unswigemteam/)
[@unsw\_igem](https://www.instagram.com/unsw_igem/)