Difference between revisions of "Team:Macquarie Australia/Notebook"

Revision as of 11:33, 9 October 2018

We went through lab safety induction to ensure safety procedures were followed at all times in the lab
We learnt how to perform digestion, ligation, transformation and how to make and run agarose gels
We used digestion to screen the Photosystem II (PSII) parts DCA, psbELJTB and psbMZtlwkOPQR. psbELJTB and psbMZtlwkOPQR showed successful digestion and correct sizes. DCA showed signs of contamination in all samples.
We decided to focus of the Chlorophyll plasmid rather than PS II. We proceeded with digestion and ligation of ChlD with ChlI2 as well as DRV1 with ChlG, both into Kanamycin backbones, following the 3A assembly protocol.
Renee, Areti and Karl also began the interlab study:

Made LB media + cam plates
Transformed Εscherichia coli DH5α cells with the interlab testing devices.Transformation was performed twice, due to poor colony growth in the LB + cam media at the first round of transformations.

We transformed the DVR1-ChlG and ChlD-ChlI2 ligation samples into competent DH5α E.coli cells. DVR1-ChlG had successful transformants which were then incubated in LB broth + Kan. ChlD-ChlI2 gave an unsuccessful transformation, so the process was repeated successfully this time. The samples were then liquid cultured.
Interlab experiments were completed this week:

Calibrations 1, 2 and 3 were completed early on in the week
Cell measurements (Abs600 and fluorescence) were taken as per the interlab protocol
Colony forming units per OD600 = 0.1 of the negative and positive control devices was also determined (in triplicates)

Set up backbone insertions for trc-POR, trc-ChlI1 (Digestion, ligation and transformation
Interlab experiments:

Colony forming units per OD600 = 0.1 of the negative and positive control devices was completed again.
Data analysis and submission.

The liquid cultures of DVR1-ChlG and ChlD-ChlI2 were miniprepped and digested (single, double digest) to screen for successful ligations by agarose gel electrophoresis (1% agarose).

The gel showed unsuccessful ligations so we are switched our ligation technique to standard assembly and backbone swapping of necessary parts

We backbone swapped DVR1 to Kan and were in the process of swapping ChlD, trc-POR and GUN4
Made up more plasmid stock of current parts by plasmid transformation, culturing and miniprep
Begun Standard assembly of DVR1 + ChlG

Ran GUN4, ChlD and trc-POR backbone swaps as well as DVR1-ChlG on a 1% agarose gel
Sent successful samples of each off for sequencing
We inserted synthesised DNA biobricks into Amp and Cam backbones for trc-POR,YidC and trc-ChlI1, trc-ycf39-HliD
We then transformed, liquid cultured, miniprepped and screened on a 1% agarose gel
We had successes for trc-ChlI1 in Amp which we sent for sequencing confirmation
Used standard assembly to assemble:

We sent CTH1-ycf54-ChlM-ter-trc-FNR-fdx as well as CTH1-ycf54-ChlM-ter-trc-FNR for sequencing
Sequencing came back for trc-ChlH to show that the promoter swap was unsuccessful. A reattempt of promoter swap is now underway
Transformed trc-POR-ChlP and ChlI2-ChlD

Irene and Renee presented at Macquarie University’s Open Day as part of the human outreach activity and spoke about iGEM and synthetic biology to potential medical science and biology students.
A new logo was created for our team.
The human practices team spoke to Dr. Paul Jaschke about the process of customer discovery and how we can potentially use it to interview for human practices and to create a customer discovery tool kit. He also arranged a meeting for us with Lynne Teo, who has worked with and explained customer discovery previously.

Irene, Nick and Renee took part in an online virtual conference ran by the iGEM Abu Dhabi team. 6 different iGEM teams met up and quickly spoke about their project for 5 minutes or so and asked questions to help other teams with their progress.
The abstract for our project was created.
Interviewed Dr Lisa Goldsmith who provided a key insight into protein purification techniques and issues that arise. Additionally she provided the team with feedback on our project and the potential impact of our project.

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