iGEM 2018 — Team UNSW_Australia

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# Team:UNSW Australia/Notebook

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Navigation

[Home](https://2018.igem.org/Team%3AUNSW_Australia)

Project

[Description](https://2018.igem.org/Team%3AUNSW_Australia/Description)
[Design](https://2018.igem.org/Team%3AUNSW_Australia/Design)
[Experimental Protocols](https://2018.igem.org/Team%3AUNSW_Australia/Experiments)
[Lab Overview](https://2018.igem.org/Team%3AUNSW_Australia/Lab)
[Cloning](https://2018.igem.org/Team%3AUNSW_Australia/Lab/Cloning)
[Protein Production](https://2018.igem.org/Team%3AUNSW_Australia/Lab/Protein)
[Assembly](https://2018.igem.org/Team%3AUNSW_Australia/Lab/Assembly)
[FRET](https://2018.igem.org/Team%3AUNSW_Australia/Lab/FRET)
[Enzyme Assays](https://2018.igem.org/Team%3AUNSW_Australia/Lab/Assays)
[Plants](https://2018.igem.org/Team%3AUNSW_Australia/Lab/Plants)
[Results](https://2018.igem.org/Team%3AUNSW_Australia/Results)
[Interlab](https://2018.igem.org/Team%3AUNSW_Australia/InterLab)

Modelling

[Overview](https://2018.igem.org/Team%3AUNSW_Australia/Model)
[Enzyme Kinetics & Diffusion](https://2018.igem.org/Team%3AUNSW_Australia/Model/EKD)
[Molecular Dynamics](https://2018.igem.org/Team%3AUNSW_Australia/Model/MD)

Parts

[Overview](https://2018.igem.org/Team%3AUNSW_Australia/Parts)
[Basic Part](https://2018.igem.org/Team%3AUNSW_Australia/Basic_Part)
[Improved Part](https://2018.igem.org/Team%3AUNSW_Australia/Improve)
[Software](https://2018.igem.org/Team%3AUNSW_Australia/Software)

Human Practices

[Overview](https://2018.igem.org/Team%3AUNSW_Australia/Human_Practices)
[Commercialisation](https://2018.igem.org/Team%3AUNSW_Australia/Human_Practices/Commercialisation)
[Education & Engagement](https://2018.igem.org/Team%3AUNSW_Australia/Public_Engagement)
[Law & Regulation](https://2018.igem.org/Team%3AUNSW_Australia/Human_Practices/Law)
[Integration](https://2018.igem.org/Team%3AUNSW_Australia/Human_Practices/Integrate)

People

[Team](https://2018.igem.org/Team%3AUNSW_Australia/Team)
[Collaborations](https://2018.igem.org/Team%3AUNSW_Australia/Collaborations)
[Attributions](https://2018.igem.org/Team%3AUNSW_Australia/Attributions)
[Experts](https://2018.igem.org/Team%3AUNSW_Australia/Journal)

Documentation

[Notebook](https://2018.igem.org/Team%3AUNSW_Australia/Notebook)
[Safety](https://2018.igem.org/Team%3AUNSW_Australia/Safety)

Judging

[Medals](https://2018.igem.org/Team%3AUNSW_Australia/Medals)
[Judging Form](https://igem.org/2018_Judging_Form?id=2710)

© 2018 UNSW Australia iGEM

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![](https://static.igem.org/mediawiki/2018/6/66/T--UNSW_Australia--Top.png)

![](https://static.igem.org/mediawiki/2018/1/16/T--UNSW_Australia--Icon-docs.png)

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# Notebook

All aspects of our lab work were documented using Benchling, and we have displayed the relevant information on this page.

Click on the buttons below to toggle the lab notes for the respective sections.

All

Cloning

Protein

Enzyme Assays

Plants

Parts

Interlab

### Week 1

#### 30th April - 6th May

#### Cloning

* Luria Broth (LB) media was made and antibiotic plates were poured out (ampicillin and kanamycin).
* A small scale grow up of NEB Turbo colonies was prepared by Dr Daniel Lorenz Winter. The cultures were miniprepped and the concentrations of the plasmids were recorded.
* Homemade Gibson Mastermix was made.

### Week 2

#### 7th May - 13th May

#### Cloning

* pETDuet-1 and pRSFDUet-1 plasmids were PCR linearised and a DpnI digest was performed. An agarose el was ran to check band sizes.

### Week 3

#### 14th May - 20th May

#### Cloning

* Gibson cloning was performed on aPFD, aPFD-SpyC, bPFD, bPFD-SnpC, IaaM-SnpT, IaaH-SpyT and mVenus-SpyT. Recombinant plasmids were transformed into cells, colony PCR (cPCR) was performed and the corresponding colonies were grown and miniprepped.
* Gel was unsuccessful.

### Week 4

#### 21st May - 27th May

#### Cloning

* cPCR of 5 more colonies were performed on the plates from Week 3.
* Gibson Assembly was performed again on the 8 constructs, cPCR was performed and agarose gel was ran. Successful transformants were grown up.

### Week 5

#### 28th May - 4th June

#### Cloning

* Overnight cultures of bacteria were miniprepped and DNA concentrations were recorded.
* Experiencing issues with cloning.
* Several controls were tested (NEB, homemade and Daniel Winter’s GIbson Assembly Mastermix).

### Week 6

#### 5th June - 10th June

#### Cloning

* pETDuet-1 and pRSFDuet-1 plasmids were PCR cleaned up and a nanodrop was performed to check DNA concentrations.
* Several more experiments were performed to identify the problem with our cloning experiments. Plasmid backbones, mastermix and DNA inserts were tested and results indicated that it may have been an issue with the Gibson Mastermix.
* 8 control plates were transformed.
* cPCR was performed on the 8 plates.

#### Enzyme Assays

* Salkowski Assay was set up to detect the end-product, Indole Acetic Acid.
* Reagents and safety documents were prepared.
* The Salkowski assay was undertaken and absorbance values recorded using a 96 well plate reader. A linear standard curve was generated.

### Week 7

#### 11th June - 17th June

#### Cloning

* Competent DH5-alpha and T7 cells were made.

### Week 8

#### 18th June - 24th June

#### Cloning

* IaaH-SpyT and IaaM-SnpT were cloned into pRSFDuet-1 plasmids and transformed into competent cells.

#### Enzyme Assay

* A standard curve for the Salkowski assay was produced with the microtitre plate (250uL volume). The Salkowski reagent and the IaaH stock was used at a 1:2 ratio.

### Week 9

#### 25th June - 1st July

#### Cloning

* cPCR was performed on the two plates (48 reactions). Colonies were grown up (however due to a mix up of gel images, the wrong IaaH-SpyT colonies were grown up).
* Overnight cultures were miniprepped and sent for sequencing. New Gibson Assembly mastermix was made.
* aPFD-SpyT and bPFD-SnpC were cloned into pETDuet-1 and transformed into cells.
* cPCR was performed on the transformed cells, and small scale grow up was made. Overnight cultures were made and gel was ran on digested products. bPFD-SnpC transformants were observed, however we were unable to detect presence of aPFD-SpyC insert.

### Week 10

#### 2nd July - 8th July

#### Cloning

* Linear pETDuet-1 and pRSFDuet-1 plasmid backbones were prepared.
* Gibson Assembly was performed on aPFD-SpyC and aPFD again. Transformation was unsuccessful.
* IaaM-SnpT plasmids were sent for sequencing.
* aPFD, aPFD-SpyC and pET linear/circular controls were transformed into DH5-alpha cells.
* Sequencing results for IaaM-Snp were successful.
* cPCR (48 reactions ) were performed on the aPFD and aPFD-SpyC plates. Successful colonies were grown up. A miniprep was performed on the overnight cultures, and a double digest was performed to verify presence of the insert by running an agarose gel electrophoresis.

#### Interlab

* Chloramphenicol plates were poured out.

### Week 11

#### 9th July - 15th July

#### Cloning

* IaaH-SpyT and bPFD-SnpC were cloned into respective plasmid vectors and transformed into cells.
* cPCR was performed (48 reactions) and successful bPFD-SnpC transformants were grown up. None of the bands were at the expected size for the IaaH-SpyT cPCR products.
* A miniprep and digest was performed on bPFD-SnpC, however none of the samples displayed a band at the right size.

#### Protein

* aPFD and aPFD-SpyC plasmids were transformed into T7 expression cells.

#### Interlab

* Interlab plasmids were transformed into DH5-alpha cells and grown up.
* Calibration assays were performed.

### Week 12

#### 16th July - 22nd July

#### Cloning

* IaaH-SpyT and bPFD-SnpC were cloned into respective plasmids and transformed into cells.
* cPCR was performed on and successful bPFD-SnpC transformants were grown up. IaaH-SpyT colonies were unsuccessful.
* Overnight cultures of bPFD-Snp were miniprepped and digested. cPCR was performed on 48 colonies from the old IaaH-SpyT plates.
* bPFD-SnpC were sent for sequencing.

#### Protein

* aPFD and aPFD-SpyC colonies were grown up and lysed with BugBuster reagent. A SDS page gel was ran on the lysed products.

#### Plants

* ½ MS agarose was made and plates were poured out.
* IAA solution was made and filter sterilised.
* Seeds were sterilised and placed onto the plates with varying concentrations of indole acetic acid (IAA).
* Plates were left for incubation

#### Interlab

* DNA from wells 2H, 2L and 2N were transformed again according to iGEM protocol.
* CFU were counted and cell growth assays were performed on the colonies, as well as the positive and negative controls. Absorbances of 1:10 dilution grow ups were measured at 600 nm.

### Week 13

#### 23rd July - 29th July

#### Cloning

* pETDuet-1 and pRSFDuet-1 plasmids were linearised.
* cPCR was performed on IaaH-SpyT. Unsuccessful PCR so nothing was grown up.

#### Protein

* IaaM-SnoopT and bPFD plasmids were transformed into T7 cells. Colonies were grown up with positive and negative controls and the cells were harvested.

#### Interlab

* 2 x grow up of each test device, final OD measurements taken for 1:10 dilution test cultures.

### Week 14

#### 30th July - 5th August

#### Protein

* The total cell protein was extracted and products were treated with BugBuster reagent. An SDS-Page gel and Western Blot was ran, however no results were obtained from these experiments. The MES buffer precipitated in the fridge.

### Week 15

#### 6th August - 12th August

#### Cloning

* mVenus-SpyT and mCerulean-SnpT were cloned into pRSFDuet-1 plasmids and transformed into cells.
* cPCR was performed on the transformed colonies (24 reactions) and a gel was ran. Successful colonies were grown up.
* The overnight mVenus and mCerulean cultures were miniprepped and a digest was performed. Gel was ran to check for the presence of the insert.

#### Protein

* IaaM-SnpT, aPFD, bPFD, bPFD-SnpC, aPFD-SpyC were transformed and expression experiments were attempted. However, the OD of the cultures were too low.
* Total cell protein was extracted and an SDS page gel was ran on the products - however there was no expression, including the controls.

#### Parts

* Double digested aPFD-SpyC and bPFD-SnoopC cloned into pET-Duet plasmids. Also digested linear pSB1C3 backbone provided by iGEM.
* We attempted to gel extract the digested inserts using a Qiagen kit however the yield was very low so we did not proceed with ligation.

### Week 16

#### 13th August - 19th August

#### Cloning

* mCerulean-SnpT and mVenus-SpyT plasmids were sent for sequencing.
* Another attempt was made to clone IaaH-SpyT into pRSFDuet-1. Gibson assembly was performed and the products were transformed into cells.
* cPCR was performed (24 reactions) and successful transformants were grown up.

#### Protein

* An attempt was made to transform and express mVenus and mCerulean 3. The cultures were lysed with BugBuster and a gel was ran on the products. We were unable to obtain expression from either parts.
* The constitutive BioBrick parts with IaaM and IaaH under the same promoter were expressed.
* Large scale expressions of aPFD, bPFD and Kinase (control) were purified, however we were unable to obtain any positive results from this experiment.

#### Plants

* Prepared ½ MS media with 1% sucrose. Surface sterilised *Arabidopsis thaliana*seeds added to 250ml of media and stored at 4C to stratify for 3 days. Remaining 250ml media prepared with 1% agar, square plates prepared.

#### Enzyme Assays

* Another Salkowski standard curve was produced. Dilutions were done in 5% EtOH.

### Week 17

#### 20th August - 26th August

#### Cloning

* Overnight cultures were miniprepped and digested. Gel was ran to check for the presence of IaaH-SpyT insert.

#### Protein

* All of the cloned plasmids were transformed into T7 and commercial Lemo cells. Glycerol stocks were made for all the parts, and were grown up and induced with IPTG. There was a lack of difference between induced and non induced cultures.

#### Plants

* Liquid culture removed from fridge, seed left illuminated at room temperature to germinate.
* 1L of 1/2 MS (1% sucrose, 1% agar) autoclaved however autoclave experienced error mid-run. Once media was able to be collected it had gone bad.
* 10mM filter sterilised IAA stock prepared.

### Week 18

#### 27th August - 2nd September

#### Cloning

* IaaH-SpyT plasmids were sent for sequencing.
* We experienced difficulties in protein expression, which may be due to the presence of the iGEM restriction sites preventing successful translation.
* Original DNA constructs were PCR modified with primers that cut out the iGEM prefix and suffix.

#### Protein

* His-mCerulean3 and His-mVenus constructs from Dr Dominic Glovers lab, His-mCerulean3-SnpT and Iaa BioBrick parts were grown up and expressed, however expression failed. This was reattempted, however results did not change.

#### Enzyme Assays

* HPLC standards made - Trp, IAA, IAM.

### Week 19

#### 3rd September - 9th September

#### Cloning

* All modified DNA constructs were cloned into corresponding plasmids and transformed into cells.
* cPCR yielded unsuccessful transformation results.
* We attempted to clone modified aPFD and bPFD into pETDUet-1. After transformation, cPCR was performed; however we were unable to detect successful transformants.
* Plasmids were PCR linearised according to protocol.

#### Protein

* mVenus and mCerulean3 were successfully expressed and purified.
* Another attempt was made to express aPFD, bPFD, mCerulean3 and mVenus, however there was no expression when the products were ran on an SDS page gel.

### Week 20

#### 10th September - 16th September

#### Cloning

* Another attempt was made in cloning aPFD and bPDS into pETDuet-1. No colonies were formed after transformation.
* Another attempt was made using commercial cells from NEB, which yielded several colonies on both plates.
* cPCR results did not yield positive results.

#### Protein

* The columns were cleaned.
* Another attempt was made to express IaaM, aPFD-SpyC and bPFD-SnpC, mVenus (positive control). Unfortunately, expression was not achieved.

#### Parts

* Phusion PCR amplification of aPFD-SpyC, bPFD-SnoopC and IaaM-SnoopT Gblocks (provided by IDT) performed.
* However primers were diluted to 100uM rather than 10uM so the PCR amplification was unsuccessful. Performed PCR amplification of Gblocks again, however extra bands on gel indicated non-specific binding of primers. We decided to generate inserts by restriction enzyme digest of already cloned pET-Duet and pRSF plasmids in future attempts.

### Week 21

#### 17th September - 23rd September

#### Cloning

* We made a decision to switch plasmid vectors for our cloning experiments, using pET-19b instead of pETDuet-1/pRSFDuet-1. DNA constructs were PCR amplified using a new set of primers, which would allow the inserts to be cloned into the new vector.

#### Protein

* An attempt was made to express aPFD and bPFD, however no expression was achieved.
* The constitutive IaaM and IaaH BioBrick was expressed.
* Waiting for the DNA constructs to be cloned into pET-19b plasmid vector.

#### Plants

* As an MS media alternative, water agar plates prepared with boiled tap water (tap water to utilise trace minerals, boiled to remove chlorine) and 1% agar.
* Water agar plates were poured out with varying concentrations of IAA and stored at -20 degrees.

#### Parts

* Double digest of aPFD-SpyC, bPFD-SnoopC and IaaM-Snoop T inserts and linearised pSB1C3 backbone vector with EcoRI-HF and PstI. After digest confirmation via an agarose gel, inserts were ligated with the pSB1C3 vector overnight.
* Ligations were transformed into DH5a cells. Transformation using our own attempts to make competent cells failed so were repeated with commercial competent cells.
* Following cPCR, successful ligations were grown up overnight and miniprepped. Following a diagnostic digest and gel, plasmids with the right insert size were sent for sequencing.

### Week 22

#### 24th September - 30th September

#### Cloning

* aPFD, bPFD, IaaH-SpyT and IaaM-SnpT were cloned into pET-19b plasmid vector and transformed into cells. cPCR was performed and successful transformants were grown up.
* aPFD, bPFD and IaaH-SpyT were successfully cloned into the new vector.
* IaaM-SnpT was PCR amplified again, and the products of the PCR were verified on a gel.
* The insert was cloned into pET-19b and transformed into cells.
* cPCR was performed on the colonies, however the results of the gel was not successful.

#### Protein

* gPFD-SpyC and gPFD with the double SpyCatcher proteins were expressed. gPFD-2xSpyCacher was successfully purified.
* aPFD, bPFD, IaaH-SpyT, gPFD-SpyC and the IaaM+IaaH BioBrick proteins were successfully expressed.

#### Plants

* Sterilised seeds were added to all the plates. Growth was observed over the next couple of weeks.

#### Parts

* Transformed IaaH BioBrick part (plate 7 W19GI) into DH5a.
* Double digest of aPFD, bPFD, IaaH-SpyT and linear pSB1C3 backbone, verified on a gel. Digests were ligated and transformed into DH5a cells.
* Sequencing results confirmed aPFD-SpyC, bPFD-SnoopC and IaaM-SnoopT were successfully cloned into the pSB1C3 vector.

#### Enzyme Assays

* An assay was performed on the cell pellets of IaaM and IaaH cultures and different time points. Unable to produce positive result.

### Week 23

#### 1st October - 6th October

#### Cloning

* New Gibson Assembly mastermix was made and IaaM-SnpT cloning was reattempted. The recombinant plasmid was transformed into cells, however no colonies were yielded.
* IaaM-SnpT recombinant plasmid was re-transformed into competent commercial cells.
* aPFD-SpyC, bPFD-SnpC, IaaH and IaaM, were to be PCR amplified using new primers for Gibson Assembly cloning into pET-19b.
* A gel was ran on the products to verify that the inserts were successfully amplified.
* The inserts (along with mVenus and mCerulean 3, kindly supplied by Django) were cloned into pET-19b and transformed into cells.
* aPFD-SpyC, bPFD-SnpC, IaaH, IaaM and IaaM-SnpT were successfully transformed onto plates. cPCR was performed, and successful transformants were grown up.
* Miniprep was performed on the overnight cultures and double digested with EcoRI and XbaI restriction enzymes.

#### Protein

* aPFD, bPFD and IaaH-SpyT cells were sonicated and the proteins were buffer exchanged into PBS. However, the proteins aggregated and crashed.
* Attempted to produce a Bradfords standard curve, however it was unsuccessful.
* gPFD-SpyC protein was purified and buffer exchanged into PBS at pH8.
* Another attempt was made to express the IaaH+IaaM BioBrick.
* The Catcher-Tag chemistry experiment was performed and given to Dr Daniel Lorenz Winter for TEM grid staining.

#### Plants

* Liquid cultures were trialed where the plants were grown in falcon tubes in MS liquid media.
* Tubes were prepared with varying concentrations of IAA (at the lower end of the concentration spectrum.
* Final growth was recorded a week after.

#### Parts

* Following cPCR of ligation transformants, successful ligations were grown up overnight and miniprepped.
* After confirmation with a diagnostic digestion and gel aPFD and bPFD selected for sequencing. IaaH-SpyC ligation unsuccessful. Another double digest of IaaH performed overnight was unsuccessful, performed two more times to obtain gel-verified digested IaaH-SpyC insert. Digest cleaned-up and ligated with linear pSB1C3 backbone. Transformed into DH5a and putative successful ligations chosen by cPCR and grown up overnight.

#### Enzyme Assays

* Another assay was performed on the cell pellets as well as the supernatant. The assay of the supernatant was successful.
* Another Salkowski assay was performed on the cell pellets and supernatants.

### Week 24

#### 8th October - 14th October

#### Cloning

* A gel was ran to verify the presence of the inserts, and successful recombinant pET-19b plasmids were sent for sequencing.

#### Protein

* Daniel performed TEM on the proteins however the staining was unsuccessful.
* A BCA assay was performed on aPFD and bPFD to determine protein concentration.
* Catcher-Tag experiment was performed with gPFD and IaaH-SpyT.
* aPFD-SpyC, bPFD-SnpC, IaaM, IaaH and IaaM-SnpT were expressed and purified.
* A BCA assay was performed on all the purified proteins.
* Sec assembly was performed.
* FRET experiments were performed with the negative controls.
* aPFD-SpyC + IaaH-SpyT assembly.
* aPFD-SpyC and IaaH-SpyT assembly experiments were performed.

#### Plants

* Final plant measurements were taken.

#### Parts

* Overnight Dh5a *E. coli* cultures containing putative pSB1C3 plasmid containing IaaH-SpyT were miniprepped and verified with diagnostic double digest and gel. Subsequently aPFD, bPFD and IaaH-SpyT in pSB1C3 constructs were sent for sequencing. Sequencing results confirmed successful cloning of aPFD, bPFD and IaaH-SpyT into pSB1C3 vector.
* All BioBrick parts dried down in iGEM provided submission plate overnight in drying oven, Sealed with foil and sent to iGEM registry. Final parts sent for submission: aPFD, bPFD, aPFD-SpyC, bPFD-SnoopC, IaaH-SpyT, IaaM-SnoopT.

#### Enzyme Assays

* Attempt to do salkowski with enzyme.
* A final attempt was made to perform the Salkowski assay.

### Week 25

#### 15th October - 21st October

#### Cloning

* Final gel depicting all 8 digested pETDuet-1 and pRSFDuet-1 plasmids was ran.

#### Protein

* Final TEM imaging by Dr Daniel Winter was performed.

#### Parts

* Double digest of all plasmid samples sent for submission performed and run on final gel to create image for wiki.


[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:UNSW Australia/Experiments

[![](https://static.igem.org/mediawiki/2018/b/b6/T--UNSW_Australia--Logo.png)](https://2018.igem.org/Team%3AUNSW_Australia)

[ ]

Navigation

[Home](https://2018.igem.org/Team%3AUNSW_Australia)

Project

[Description](https://2018.igem.org/Team%3AUNSW_Australia/Description)
[Design](https://2018.igem.org/Team%3AUNSW_Australia/Design)
[Experimental Protocols](https://2018.igem.org/Team%3AUNSW_Australia/Experiments)
[Lab Overview](https://2018.igem.org/Team%3AUNSW_Australia/Lab)
[Cloning](https://2018.igem.org/Team%3AUNSW_Australia/Lab/Cloning)
[Protein Production](https://2018.igem.org/Team%3AUNSW_Australia/Lab/Protein)
[Assembly](https://2018.igem.org/Team%3AUNSW_Australia/Lab/Assembly)
[FRET](https://2018.igem.org/Team%3AUNSW_Australia/Lab/FRET)
[Enzyme Assays](https://2018.igem.org/Team%3AUNSW_Australia/Lab/Assays)
[Plants](https://2018.igem.org/Team%3AUNSW_Australia/Lab/Plants)
[Results](https://2018.igem.org/Team%3AUNSW_Australia/Results)
[Interlab](https://2018.igem.org/Team%3AUNSW_Australia/InterLab)

Modelling

[Overview](https://2018.igem.org/Team%3AUNSW_Australia/Model)
[Enzyme Kinetics & Diffusion](https://2018.igem.org/Team%3AUNSW_Australia/Model/EKD)
[Molecular Dynamics](https://2018.igem.org/Team%3AUNSW_Australia/Model/MD)

Parts

[Overview](https://2018.igem.org/Team%3AUNSW_Australia/Parts)
[Basic Part](https://2018.igem.org/Team%3AUNSW_Australia/Basic_Part)
[Improved Part](https://2018.igem.org/Team%3AUNSW_Australia/Improve)
[Software](https://2018.igem.org/Team%3AUNSW_Australia/Software)

Human Practices

[Overview](https://2018.igem.org/Team%3AUNSW_Australia/Human_Practices)
[Commercialisation](https://2018.igem.org/Team%3AUNSW_Australia/Human_Practices/Commercialisation)
[Education & Engagement](https://2018.igem.org/Team%3AUNSW_Australia/Public_Engagement)
[Law & Regulation](https://2018.igem.org/Team%3AUNSW_Australia/Human_Practices/Law)
[Integration](https://2018.igem.org/Team%3AUNSW_Australia/Human_Practices/Integrate)

People

[Team](https://2018.igem.org/Team%3AUNSW_Australia/Team)
[Collaborations](https://2018.igem.org/Team%3AUNSW_Australia/Collaborations)
[Attributions](https://2018.igem.org/Team%3AUNSW_Australia/Attributions)
[Experts](https://2018.igem.org/Team%3AUNSW_Australia/Journal)

Documentation

[Notebook](https://2018.igem.org/Team%3AUNSW_Australia/Notebook)
[Safety](https://2018.igem.org/Team%3AUNSW_Australia/Safety)

Judging

[Medals](https://2018.igem.org/Team%3AUNSW_Australia/Medals)
[Judging Form](https://igem.org/2018_Judging_Form?id=2710)

© 2018 UNSW Australia iGEM

![](https://static.igem.org/mediawiki/2018/6/66/T--UNSW_Australia--Top.png)

![](https://static.igem.org/mediawiki/2018/6/66/T--UNSW_Australia--Top.png)

![](https://static.igem.org/mediawiki/2018/8/8d/T--UNSW_Australia--Icon-lab.png)

![](https://static.igem.org/mediawiki/2018/f/f3/T--UNSW_Australia--Icon-lab-bw.png)

# Experiments

This page includes all the experimental protocols used by the UNSW iGEM team. Please click on each heading to view the protocols or view our weekly progress on the [notebook](https://2018.igem.org/Team%3AUNSW_Australia/Notebook) page. Alternatively, to see the detailed experimental introduction, results and discussion, please visit the [lab overview](https://2018.igem.org/Team%3AUNSW_Australia/Lab) page.

## DNA Cloning

## Linearisation pETDuet1 and pRSFDuet1 Plasmid Backbones Using PCR

1. Forward and reverse primers created for the plasmids.
2. 2 µL of plasmid + 198 µL of water.
3. PCR amplification, according to the table below:

| Component | 50 µL Reaction | Final Concentration |
| --- | --- | --- |
| Q5 High-Fidelity 2X Master Mix |

 25 µL | 1X || 10 µM Forward Primer | 2.5 µL | 0.5 µM |
| 10 µM Reverse Primer | 2.5 µL | 0.5 µM |
| Template DNA | 2 µL diluted | < 1,000 ng |
| Nuclease-Free Water | 18 µL |  |
| Step | Temperature | Time |
| --- | --- | --- |
| Initial Denaturation | 98°C | 30 seconds |
| 25–35 Cycles | 98°C | 5–10 seconds |
| 62°C | 10–30 seconds |
| 72°C | 2 minutes |
| Final Extension | 72°C | 2 minutes |
| Hold | 4–10°C |  |

## Plasmid Digest (Dpn1 Digest)

1. Set-up the reaction mixture:

   |  |  |
   | --- | --- |
   | Restriction Enzyme | 1 µL |
   | DNA | 1 µg |
   | 10X Cutsmart | 5 µL (1X) |
   | Total Reaction Volume | 50 µL |
2. Incubate for 1 hr at 37°C.
3. Heat inactivate at 80°C for 20 minutes.

## Agarose Gel Electrophoresis

1. Combine 1 g of agarose powder with 100 mL of 1x TAE buffer in a microwavable flask.
2. Microwave for 1-2 minutes until the agarose is completely dissolved. Avoid over-boiling, and stop to swirl the flask every 20-30 seconds until the solution is clear.
3. Allow the solution to cool down until you can comfortably hold the flask with your hand, then add 1 µL of RedSafe.
4. Seal the ends of a gel tray and pour the solution into the tray with a well comb in place. Let the solution sit at room temperature for 20-30 minutes until it solidifies into a gel.
5. Place the gel into the gel tank, and fill the tank with 1X TAE buffer until the gel is covered. Remove the well comb.
6. Mix 2 µL of the DNA sample with 3 µL of H2O and 1 µL of 6X loading dye.
7. Load DNA ladder into the first well of the gel, followed by your DNA samples.
8. Connect the gel tank to a power pack and run the gel at 100 V for 1 hr.
9. Carefully take the gel tray to a Gel Doc for imaging.

## Gibson Assembly

### Materials:

5X Isothermal Reaction Mix (6 mL total, Store at -20°C):

|  |  |
| --- | --- |
| 1 M Tris-HCl (pH 7.5) | 3 mL |
| 1 M MgCl2 | 300 µL |
| 100 mM dGTP | 60 µL |
| 100 mM dATP | 60 µL |
| 100 mM dTTP | 60 µL |
| 100 mM dCTP | 60 µL |
| 1 M DTT | 300 µL |
| PEG-8000 | 1.5 g |
| 100 mM NAD | 300 µL |
| H2O | 360 µL |

Assembly Master Mix (1.2 mL total, store in 15 µL aliquots at -20°C.):

|  |  |
| --- | --- |
| 5X Isothermal Master Mix | 320 µL |
| 510 U/µL T5 exonuclease | 0.64 µL |
| 2 U/µL Phusion DNA Polymerase | 20 µL |
| 40 U/µL T4 DNA Ligase | 0.16 µL |
| H2O | 860 µL |

### Method:

1. PCR amplify or digest your fragments of choice and gel purify.
2. If PCR from a methylated DNA template (e.g. linearised plasmid), a DpnI digest can be used to remove the unwanted template plasmid. Clean up afterwards.
3. Thaw a 15 µL assembly mixture aliquot and keep on ice until ready to be used.
4. Nanodrop your DNA fragments. Prepare 50-100 ng of vector with 2-3 fold excess of insert in nuclease-free water to a total volume of 5 µL.
5. Add 15 µL assembly mixture to the DNA mixture, for a total reaction volume of 20 µL.
6. Incubate at 50°C for 15 to 60 minutes (60 minutes is optimal).

## Heat Shock Transformation

1. Incubate 50 ng of plasmid construct with 25 µL of chemically competent *E. coli* T7 express or DH5α on ice for 30 minutes.
2. Heat shock the cells for 45 seconds at 42°C and place back onto ice for 2 minutes.
3. Allow cells to grow for 45 minutes in 200 µL of SOC outgrowth media (NEB) at 37°C and 200 rpm.
4. Spread plate onto Luria Broth (LB) agar plates containing appropriate antibiotics at working concentration and grow at 37°C overnight.

## Colony PCR

1. Pick up individual bacterial colonies from an agar plate that was grown overnight using a pipette tip, and dilute each colony into 50 µL of water.
2. Prepare the PCR master mix by multiplying the volume for each component shown below (for 1 reaction) by the number of colonies to sample +1 (i.e. number of reactions plus 1 excess).
   * 18 µL nuclease free water.
   * 5 µL 5X Taq master mix.
   * 0.5 µL 10 µM T7 promotor primer
   * 0.5 µL 10 µM T7 terminator primer
3. Add 1 µL of each diluted colony and 24 µL of PCR master mix to a PCR tube.
4. Run PCR with the following steps (lid at 105°C and volume = 20 µL):
   * Step 1: 95°C for 5 minutes
   * Step 2: 95°C for 30 seconds
   * Step 3: 55°C for 30 seconds
   * Step 4: 68°C for 2 minutess
   * Repeat 25-35 cycles of steps 2-4
   * Step 5: 68°C 5 minutes
   * Step 6: Hold at 4°C
5. Run PCR products on a 1 % agarose gel and image for analysis.

## Sequencing

1. Transfer 10 µL of purified plasmid sample (50-100 ng/µL) to a PCR tube. Add 5 µL of one primer (forward or reverse).
2. Request sequencing at the UNSW Centre for Genomics via the on-line portal.
3. Label PCR tubes carefully with numbers as per the on-line order.
4. Take PCR tubes to the Ramaciotti Centre for Genomics Lvl 2 (at UNSW, Sydney, Australia), and store the samples in the fridge provided.
5. Sanger sequencing is carried out by the Centre staff following [the provided protocol](http://www.ramaciotti.unsw.edu.au/wp-content/uploads/2016/09/RAMAC_LIMS_User_Guide-2016.pdf).

## Restriction Cloning

1. Set-up the reaction mixture:

   |  |  |
   | --- | --- |
   | Restriction Enzyme | 1 µL of each enzyme |
   | DNA | 1 µg |
   | 10X Cutsmart | 5 µL (1X) |
   | Total Reaction Volume | 50 µL |
2. Incubate for 1 hr at 37°C.
3. Heat inactivate at 80°C for 20 minutes.

### Ligation

1. Set up the following reaction in a microcentrifuge tube on ice:

   | Component | 20 µL Reaction |
   | --- | --- |
   | T4 DNA Ligase Buffer (10X)\* | 2 µL |
   | Vector DNA | 50 ng |
   | Insert DNA | A molar ratio of 1:3 vector to insert should be used |
   | Nuclease-free water | to 20 µL |
   | T4 DNA Ligase | 1 µL |
2. Gently mix the reaction by pipetting up and down and microfuge briefly.
3. For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.
4. Heat inactivate at 65°C for 10 minutes.
5. Chill on ice and transform 1-5 µL of the reaction into 25 µL competent cells.

## Miniprep

Protocols were followed from the Qiagen QIAprep Spin Miniprep Kit Cat No./ID: 27104. No changes were made.

## Protein Production

## Starter Culture

One colony was selected from the plate grown overnight and grown in 2 mL of LB containing appropriate antibiotic at 37°C and 200 rpm overnight.

## Large-scale grow-up

1. Baffled shake flasks containing 500 mL of LB with 50uL of the appropriate antibiotic at 37°C are inoculated with the starter culture.
2. The cells are grown at 37°C and 200 rpm and OD600 is periodically measured.
3. Once OD600 reaches an absorbance reading of 0.6, add 1mM of IPTG to induce expression of the protein.
4. After induction, grow the cells overnight at 24°C, 200 rpm.

## Collection of Cells by Centrifugation

1. Centrifuge the bacterial culture at 4600 x g for 20 minutes.
2. Collect cell pellet and resuspended in binding buffer (20 mM NaH2PO4, 500 mM NaCl, 10 mM Imidazole).

## Cell Lysis by Sonication

1. Lyse the cell pellet by sonication (Branson) for 10 minutes at 50% amplitude, alternating 2 second intervals, kept on ice.
2. Centrifuge the cell lysate at 15000 rpm for 45 minutes.
3. Collect the supernatant (soluble fraction).

## IMAC

Immobilised Metal ion Affinity Chromatography (IMAC) was performed to purify the expressed proteins.

1. His-tagged protein is bound to a 1 mL Ni-NTA Superflow Cartridge (Qiagen) by loading the soluble fraction of the cell lysate onto the column.
2. Wash with 10 mL of binding buffer (20 mM NaH2PO4, 500 mM NaCl, 10 mM Imidazole).
3. Elute with 2 mL of elution buffer (20 mM NaH2PO4, 500 mM NaCl, 500 mM Imidazole).
4. Analyse fractions with SDS-PAGE.

## Buffer Exchange

### Column

1. Elutions were analysed with SDS-PAGE and buffer exchanged into PBS (pH 8) using Pierce Protein Concentrators PES, 10K MWCO, 2-6 mL (Thermo Scientific).
2. Add protein to the column.
3. Top up column to 6 mL with PBS buffer.
4. Centrifuge column at 4600 x g for 20 minutes.
5. Repeatedly centrifuge, discard flow through, and top up with PBS buffer (pH 8) until dilution factor of 0.01 is reached. That is, until there is 1% of the old buffer left in the solution.

### Dialysis

1. Add 1 mL of protein and 1 mL of PBS buffer (pH 8) to a 15 mL Falcon tube.
2. Add 2 mL of the solution to a SnakeSkin™ Dialysis Tubing, 10K MWCO, 22 mm.
3. Use dialysis tubing clamps (one-piece polypropylene clamp) to further secure the solution inside the SnakeSkin dialysis tubing.
4. Add 500 mL of PBS buffer, pH 8, (this is the buffer we want to exchange into) into a 500 mL glass beaker.
5. Place the dialysis tubing with the solution into the beaker.
6. Place the beaker on top of a magnetic stirrer, 75 rpm, and leave overnight.

## Western Blot

### Materials

* NuPAGE Bis-Tris gel
* NuPAGE MES running buffer
* Mini iBlotTM stack
* TBS-T:
* 1X TBS with 0.1 % Tween20
* Blocking Solution:
* 5 % skim milk in TBS-T
* Antibody Solution:
* 1:2000 dilution of HRP conjugated anti-His-tag antibody in TBS-T + 1 % BSA
* Chemiluminescent HRP substrate

### Sample Preparation

1. Add reducing buffer to the bacterial lysates.
2. Heat at 95°C for 5 minutes.

### SDS-Page Gel

1. Remove the NuPAGE gel from its packaging and peel off the plastic strip from its base.
2. Place the gel inside the tank, and fill with NuPAGE MES running buffer.
3. Load 5 µL of the protein standards ladder into the first well.
4. Load up to 20 µL of each lysate sample into the wells.
5. Connect the gel tank to a power pack, and run at 160 V for 40 minutes.

### Protein Transfer

1. Remove and rinse the gel in water.
2. Inside an iBlot Transfer Device, assemble the mini stack with the gel inside.
3. Run at 20 V for 7 minutes.

### Blocking

1. Incubate the membrane for 1-2 hours in blocking solution at room temperature, shaking.

### Antibody Staining

1. Incubate the membrane in antibody solution either at 4°C overnight, or at room temperature for 2 hours.
2. Wash the membrane in TBS-T three times for 10 minutes per wash at room temperature, shaking.

### Detection

1. Remove membrane from the last wash and place in chemiluminescent image analyser.
2. Prepare HRP substrate according to manufacturer's instructions and add to the membrane.
3. Image.

## Assembly

## Catcher and Tag Assembly

1. Purified IaaH fused to SpyTag (IaaH-SpyTag) and prefoldin proteins fused to SpyCatcher (aPFD-SpyCatcher, gPFD-SpyCatcher and SpyCatcher-gPFD-SpyCatcher) were obtained.
2. IaaH-SpyTag was mixed with either aPFD-SpyCatcher, gPFD-SpyCatcher and SpyCatcher-gPFD-SpyCatcher at a concentration of 3 µM and 15 µM respectively in a total volume of 250 µL in PBS pH 8, and incubated at room temperature.
3. After 0, 10, 20 and 30 minutes of incubation, a 10 µL sample was obtained.
4. 5 µL of 4X Bolt LDS sample buffer was added to each sample, and then boiled for 10 minutes at 95°C to cease SpyCatcher reactivity whilst preserving any covalent interactions.
5. The samples were then examined via SDS-PAGE.

## Size Exclusion Chromotography (SEC) We would like to thank Hélène Lebhar of the UNSW Recombinant Proteins facility for conducting SEC experiments for us.

## TEM

### Materials

* Protein sample (ideally in water or low salt buffer)
* Milli-Q water
* 2 % uranyl acetate
* Parafilm
* Self-closing forceps

### Staining

1. Glow discharge the carbon-coated grids (carbon-coated side up) to render them hydrophilic.
2. Place the grids (carbon-coated side up) on parafilm.
3. Place 7 µL of sample on a grid.
4. Let the grid stain for 10 minutes.
5. Using a pasteur pipette, transfer 5 droplets of Milli-Q water on a piece parafilm (for each grid).
6. After the 10 minutes of staining, pick up grid with the forceps and wash residue salts by passing the grid through the five droplets of water.
7. In a fume hood, place one droplet of 2 % uranyl acetate on a piece of parafilm (for each grid).
8. Wear appropriate PPE to handle uranium; do not work with uranium outside the fume hood.
9. Place the grid on the uranyl acetate drop (carbon-coated side down) and stain for 5-7 minutes.
10. Pick the grid up with the forceps.
11. Remove excess uranyl acetate by touching one side of the grid with filter paper for a few seconds, then briefly touch the other side of the grid with the filter paper.
12. Let the grid dry for a few minutes before transferring back it to its box.
13. The grid can be fully dried in a oven if imaging the sample the next day, or just left in the box until imaging.

## FRET

## FRET Protocol for Negative Controls

### Part 1

1. Dilute mCerulean3 (Cerulean) and mVenus (Venus) protein samples to 1 mg/mL (≈ 34 µM) with PBS pH 7.4
2. Create a serial dilution of both Cerulean and Venus from 1 mg/mL down to 1 µg/mL in an (ideally) black sided 96-well plate (200 µL of undiluted protein, transfer 20 µL to 180 µL PBS, and so on).
3. Scan the excitation and emission spectra of these wells to determine the ideal values of excitation and emission for each value, using the values from Markwardt *et al.* and Jonáš *et al.* as shown in the table as a starting point.
4. Blank appropriately with 180 µL PBS.

|  | Excitation | Emission |
| --- | --- | --- |
| mCerulean3 | 433 (400-465) | 475 (465-525) |
| mVenus | 515 (500-525) | 528 (520-550) |

The values in these tables were taken from, Markwardt *et al.* (2011)1 and Jonáš *et al.* (2014)2.

* Dilute samples equally if concentration is too high for the fluorescence reader of the machine.

### Part 2

1. Add 90 µL of 1 mg/mL protein to 90 µL PBS for each protein in triplicate.
2. Add 90 µL of Cerulean to 90 µL Venus in triplicate.
3. Blank with 180 µL PBS.
4. Excite all wells at the ideal excitation value determined for Cerulean and scan wavelengths longer than this.
5. Please see [supplementary data](https://static.igem.org/mediawiki/2018/3/35/T--UNSW_Australia--FRET2018.zip) for specific parameters used.

## Enzyme Assays

## BCA Protein Assay

Protocol followed as per Pierce BCA Protein Assay Kit 23225 to determine protein concentration in solution:

* Sample to working reagent ratio = 1:8
* 96-well plate reader: SPECTROstar Nano BMG LABTECH
* Software used: SPECTROstar Nano

## Salkowski Assay

The Salkowski assay is a measurement-based protocol used to assess the quantity of indole-3-acetic acid within a sample. This particular protocol has been adapted from the methods used by [2011 iGEM Imperial College London](https://2011.igem.org/Team%3AImperial_College_London/Project_Auxin_Design), Tang and Bonner’s 1947 paper3, and Glickmann and Dessaux's 1995 paper4.

### Cellular Protocol

1. Pick a single colony of untransformed T7 and transformed T7 and inoculate a 2-5ml overnight culture in LB.
2. Add antibiotics to the transformed culture.
3. The next day, add 50 mL of LB + 2.5 mg/mL tryptophan to a 250 mL shake flask labelled “Control”.
4. Add 50 mL of LB + 2.5 mg/mL tryptophan + antibiotic to another baffled shake flask labelled “IAA”.
5. Add 1 mL of the respective overnight cultures to each flask. Incubate with shaking (200 rpm) at 37°C. Sample at 1h, 4h, 7h and 20-24h.
6. For each sample: a. Measure the OD at 600 nm.
7. Spin down 1 mL. Retain pellet and freeze. Retain supernatant and store at 4°C. Label these 4.
8. The next day: Add Salkowski reagent to supernatants at the ratio of 1 mL per 0.5 mL of supernatant. Incubate in the dark for 30 minutes.
9. Add 1 mL of Salkowski reagent to each cell pellet.
10. Vortex to mix.
11. Incubate in the dark for 30 minutes.
12. Centrifuge at full speed for 5 minutes.
13. Measure the absorbance at 530 nm Measure appropriate controls as well.
14. Normalise readings at each time point against OD600.

The assay is performed by adding the Salkowski reagent to a prepared sample:

1. To prepare the Salkowski assay reagent, slowly add 95 % H2SO4 to Milli-Q water in a ratio of 3:2, allowing time for heat dissipation between additions.
2. Dissolve 4.5 g/L of anhydrous FeCl3 into the solution. Store in the dark, wrapped in foil.
3. Prepare the standard by using a 3 mM stock, then diluted 1 in 10 for usage – with 300 µM to 0 µM concentrations.
4. Prepare a standard curve.
5. Samples are run by adding the sample (or standard) to the Salkowski reagent in a ratio of 1:2 in a time sensitive manner within a 96-well microtitre plate.
6. Run the plate on a plate reader for absorbance at 530 nm, after being left for 30 minutes in the dark.

## High Performance Liquid Chromotography (HPLC)

1. Acidify supernatant with 5 M HCl to pH 2.5.
2. Extract supernatant with an equal volume of ethyl acetate.
3. Evaporate off the solvent by either drying it through a vacuum or dissolving in methanol & acetic acid at pH 4.5 (25 % methanol, 1 % acetic acid, made up to 1 mL with H2O).
4. 250 µL methanol, 10 µL acetic acid, 740 µL H2O.
5. Run system with solution 72 % of 1 % acetic acid and 28 % of 100 % methanol for 0.8 mL/minute with injection of 30.

## Plants

## Seed Surface Sterilisation

1. Wild-type *Arabidopsis thaliana* seeds were surface sterilised by mixing in a solution of 2.5 % sodium hypochlorite with a drop of Tween for 10 minutes.
2. Seeds were subsequently washed eight times in sterile water.

## Indole-3-acetic Acid Filter Sterilisation

1. 10 mM stock indole-3-acetic acid (IAA) was prepared by dissolving indole-3-acetic acid sodium salt purchased from Sigma Aldrich in ethanol.
2. In a laminar flow hood, the solution was filter sterilised through a 0.22 µm Millex® syringe driven filter and stored at 4°C in a foil covered container to protect from light.

## MS Agar Plate Preparation

1. Murashige and Skoog (MS) media was prepared using M5519 MS basal powdered medium purchased from Sigma Aldrich.
2. In four separate Schott bottles, 250 mL of MS media was prepared using 1.1 g of MS basal salts (per manufacturer instructions) and Milli-Q purified water.
3. Each mixture was supplemented with sucrose (1 % w/v) and agar powder (0.8 % w/v) and adjusted to pH 5.7 using KOH.
4. Mixtures were autoclaved and allowed to cool to approximately 50°C (or until able to be held comfortably).
5. Cooled bottles were transferred to a dark laminar flow hood where indole-3-aecetic acid solution was added to prepare four separate MS agar mixtures at 0 µM, 1 µM, 10 µM and 100 µM concentrations IAA respectively.
6. In a dark laminar flow hood, 50 mL of media was pipetted into a square plate using a serological pipette. Four 50 mL plates were prepared for each concentration of IAA.
7. Plates were labelled, sealed with parafilm and stored wrapped in foil at 4°C until further use.

## Liquid Culture Media Preparation

1. MS media was prepared using M5519 MS basal powdered medium purchased from Sigma Aldrich.
2. In an autoclavable Schott bottle, 500 mL 1/2 MS media was prepared using 1.1 g of MS basal salts and 500 ml of Milli-Q purified water (4.4 g salts equivalent to 1 L MS media per manufacturer’s instructions).
3. Media was supplemented with sucrose (1 % w/v) and adjusted to pH 5.7 using KOH.
4. Media was autoclaved and stored at room temperature until further use.

## *Arabidopsis thaliana* Growth on Agar Plates

1. Surface sterilised seeds were added to a flask containing an autoclaved solution of Milli-Q water with 1 % w/v sucrose in a laminar flow hood.
2. This flask was covered with foil and seeds were stratified at 4°C for two days.
3. Stratified seeds were then allowed to germinate under light at room temperature for 2 days.
4. In a dark laminar flow hood, seedlings were transplanted to MS media plates with varying concentrations of IAA. Approximately five seedlings were transplanted per plate using sterile tweezers and loops. Seedlings were arranged in a line, 2 cm from the top of each plate. Three plates with seedlings were prepared for each concentration.
5. Plates were sealed with parafilm and covered with foil leaving only the top 2 cm exposed in order to limit IAA photodegradation and simulate the natural growth conditions of higher plants whereby only shoots are exposed to illumination5. Plates were stored upright at an approximately 90° angle. The plated seedlings were grown under light at room temperature for 20 days.
6. Primary root length and lateral root numbers were measured at 4, 6, 11 and 20 days following the initial seedling transplant.

## *Arabidopsis thaliana* Liquid Culture

1. In a laminar flow hood, 25 mL of 1/2 MS media with 1 % sucrose was aliquoted into twelve 50 mL foil wrapped falcon tubes.
2. Filter sterilised IAA was added to prepare duplicate tubes at 0 nM, 1nM, 10 nM, 100 nM, 1 µM and 10 µM concentrations of IAA.
3. 100 µL of surface sterilised *A. thaliana* seeds were added to each tube.
4. Liquid cultures were stratified at 4°C for two days, then grown under light at room temperature for 9 days.
5. Three seedlings from each tube were selected at random and measured for primary root length and shoot length.

## References

1. Markwardt, M. et al. An improved cerulean fluorescent protein with enhanced brightness and reduced reversible photoswitching. *PLOS ONE* **6** e17896 (2011).
2. Jonáš, A. et al. In vitro and in vivo biolasing of fluorescent proteins suspended in liquid microdroplet cavities. *Lab Chip* **14** 3093-3100 (2014).
3. Tang, YW. and Bonner, J. The enzymatic inactivation of indoleacetic acid; some characteristics of the enzyme contained in pea seedlings. *Arch. Biochem.* **13** 11–25 (1947).
4. Glickmann, E. and Dessaux, Y. A critical examination of the specificity of the salkowski reagent for indolic compounds produced by phytopathogenic bacteria. Applied and Environmental Microbiology **61** 793–796 (1995).
5. Xu, W. et al. An improved agar-plate method for studying root growth and response of *Arabidopsis thaliana*. *Sci Rep* **3** 1273 (2013).

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# Team:UNSW Australia/Results

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# Results

## Key Findings

Our project has provided the proof of principle for a prefoldin scaffold that co-localises enzymes. We have proven that our attachment mechanisms (SpyTag/Catcher and Snoop Tag/Catcher) work between enzymes and prefoldin, and that the heterohexameric structure of alpha and beta prefoldin self-assembles. Our modelling was successful in predicting the effect of scaffolding enzymes to prefoldin, taking into consideration enzyme kinetics and molecular dynamics. Furthermore, we believe our software part will prove to be a useful search tool for future iGEM teams.

## Results by Section

![](https://static.igem.org/mediawiki/2018/0/0a/T--UNSW_Australia--Icon-cloning.png)

### Cloning

Eight DNA constructs were cloned into pETDuet-1 and pRSFDuet-1 plasmids using Gibson assembly. Six of these constructs were subcloned into pET19b for protein expression. In addition to this, BBa\_K1789000 (IaaM) and BBa\_K1789001 (IaaH) were cloned into pET19b for protein expression. All recombinant plasmids were validated by a diagnostic digest and Sanger sequencing.

![](https://static.igem.org/mediawiki/2018/c/c7/T--UNSW_Australia--Icon-protein.png)

### Protein Production

Nine proteins were expressed in *Escherichia coli* T7 cells and purified using Immobilised Metal Affinity Chromatography (IMAC). These included prefoldin proteins that form the basis of our scaffold, and prefoldin proteins fused with SpyCatcher and SnoopCatcher. Enzymes and fluorescent proteins were expressed and purified, including the enzyme indole acetamide hydrolase (IaaH) with a C-terminal SpyTag for conjugation experiments.

![](https://static.igem.org/mediawiki/2018/c/c4/T--UNSW_Australia--Icon-assembly.png)

### Assembly

The formation of our enzyme-scaffold complex requires two stages of assembly: firstly, the formation of the alpha prefoldin and beta prefoldin hexamer, and secondly the covalent attachment of enzymes to the scaffold through SpyTag/SpyCatcher or SnoopTag/SnoopCatcher reactions. We have assembled alpha and beta prefoldin hexamers and covalently attached the enzyme IaaH-SpyTag to alpha prefoldin and gamma prefoldin scaffolds. Furthermore, we visualised filaments of wild-type gamma prefoldin, gamma prefoldin-SpyCatcher, and gamma prefoldin-SpyCatcher attached to IaaH-SpyTag.

![](https://static.igem.org/mediawiki/2018/3/34/T--UNSW_Australia--Icon-fret.png)

### FRET

FRET is a method for assessing the distance between two molecules based on energy transfer between two fluorophores. To assess the distance between enzymes attached to our scaffold we planned to perform FRET using the fluorescent proteins mCerulean3 and mVenus free. FRET was successfully performed for mCerulean3 and mVenus free in solution. However, due to time constraints we were unable to express and purify mCerulean3 or mVenus fused to Spy/Snoop Tags for attachment to our scaffold and use in FRET. Nevertheless, we have obtained the optimum excitation wavelengths for mCerulean3 and mVenus and established a working FRET protocol for future use.

![](https://static.igem.org/mediawiki/2018/e/e1/T--UNSW_Australia--Icon-assay.png)

### Enzyme assays

Biosynthesis of indole-3-acetic-acid (IAA) is the two-step enzymatic reaction we selected to use as proof of concept of our Assemblase scaffold. To compare reaction efficiency in a scaffolded versus unscaffolded enzyme scenario, we successfully set-up two quantitative assays for determining the concentration of reaction intermediates and products over time. We created standard curves for these assays based on unscaffolded enzyme reactions only due to time constraints. In the future these assays can be utilised to test the effect of enzyme scaffolding to our Assemblase system on protein yield.

![](https://static.igem.org/mediawiki/2018/d/d0/T--UNSW_Australia--Icon-plants.png)

### Plants

Auxins are plant hormones involved in the regulation of plant growth and development. The biosynthesis of the auxin indole-3-aecetic acid (IAA) was used as a test pathway for our scaffold system, and thus a plant growth assay was developed to investigate the functionality of biosynthetically produced IAA. We successfully observed effects of commercially obtained IAA on plant root growth, namely increased lateral root growth and decreased primary root growth. These results are consistent with reports in the literature. Future work could further optimise the concentration of exogenous IAA added to the plant growth agar, and incorporate IAA produced through our Assemblase system.

![](https://static.igem.org/mediawiki/2018/4/44/T--UNSW_Australia--Icon-modelling.png)

### Enzyme kinetics modelling

The effect of the distance between the enzymes on the reaction rate was modelled. We found that the model predicted a 5-fold improvement in IAA production when the enzymes are clustered together using Prefoldin, compared to when they are free in solution.

![](https://static.igem.org/mediawiki/2018/4/4f/T--UNSW_Australia--Icon-MD.png)

### Molecular Dynamics

Allowed us to visualise the physical movements of our scaffold and determine key parameters for use in the mathematical model as we were able to approximate the average distance between enzymes when attached to the Prefoldin scaffold.

![](https://static.igem.org/mediawiki/2018/c/c0/T--UNSW_Australia--Icon-parts.png)

### Software

This tool was developed to provide future iGEM teams an improved tool to scan the Biobricks Database using Python 3. Along with the search tool, we provide a method for generating a clean xml file of the iGEM database as well as incorporating negative searching into the tool to allow for searching of parts that do not meet search criteria.