Team:UNSW Australia/Notebook

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

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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 thalianaseeds 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.