Team:Macquarie Australia/trial

With the break between semesters over, the Macquarie iGEM returned to classes. For us this was the first day of iGEM 2012. We had our first meeting and discussed our project. Dr. Louise Brown and Associate Professor Rob Willows were introduced the team and we were began to determine who would take on certain roles within the team.

We eagerly began our project, deciding to use the novel approach of Gibson Assemble to produce our optimised genes. We had decided to develop a gene-switch controlled by light. To do this a couple of genes would be needed:

1. a bacteriophytochrome
2. Heme oxygenase

The bacteriophytochromes from Deinococcus radiodurans and Agrobacterium tumefaciens were chosen. Over the next week Matt Stclair started to develop the G-blocks by:

1. Acquiring the DNA sequence
2. Translating into the protein sequence
3. Using the DNA sequence, optimise codon usage for E. coli
4. Translating into protein sequence and ensuring no changes in amino acids relative to the original sequence

Our lab work began today with the preparation of liquid media, plates and buffers. The protocols we followed are located here.

We prepared the following:

• Liquid LB Media
• SOC Solution
• SOB Solution
• LB Agar Plates
• Ampicillin LB Agar Plates: 31 plates
• Chloramphenicol LB Agar Plates: 33 Plates
• Kanamyacin LB Agar Plates: 32 Plates
• TB buffer.
• TAE buffer.
• EDTA buffer.

While the lab group produced the necessary materials, another team began developing the G Blocks. The G Block team consisted of Ellaina, Matt Smith, Matt Stclair, Harry, Gazelle, Silas, Kim, and Miguel. Matt Stclair spearheaded the effort and proofread all the sequences we had developed to ensure that we had not changed the protein sequence or introduced restriction sites.

Ryan volunteered to be the wiki chief with Erin helping out throughout the project. Ellaina ensured the key safety questions were answered, and relayed these to the rest of the team. Rob and Sarah took control of the seeking finances to get the team to Hong Kong.

In a Macquarie iGEM first, the team took a big interest in the idea of human outreach and we had a brainstorming session. Ellaina proposed that we visit a high school while Elle suggested that the Universities open day would be a great opportunity to communicate with the wider community. Ellaina took control of developing our high school visit and Elle took control of Open Day.

In anticipation of our G Blocks arriving next week, competent cells were prepared. The protocol followed can be seen here.

Human Practice planning went into overdrive this week. To engage the wider community, fluorescent and bioluminescent parts from the registry were selected and transformed in E. coli. Red, Green, Orange, and Cyan Florescent protein as well as Luciferase were all located and transformed in the competent cells available. More details can be seen here.

Our open day preparation did not go as planned. Most of our transformations were unsuccessful, we believe this was because SOC was added after the heat shock. As disappointing as this was, there was one little piece of good news.

A single colony of Top 10 E. coli containing GFP insert was identified on a plate of LB agar media. LB agar and nutrient agar plates were struck using this colony. The colony was also used to inoculate 5mL of nutrient broth. Plates and stock were then incubated at 37°C overnight. Both of the streaked plates and the broth showed significant growth of E. coli colonies which could fluoresce when exposed to UV light.

We got our first news on the finances front. Unfortunately, Qantas declined to sponsor the effort of getting our team to Hong Kong. This was soon forgotten when the Macquarie University School of Medicine, Agilent and the Defence Science Institute (thanks Yagiz) all agreed to sponsor our team. Hong Kong here we come.

Design of the G Blocks continued. The fragments were designed with melting temperatures NEED TO ASK STCLAIR. However, IDT had significant synthesising them due to GC rich regions. Unfortunately for the MQ iGEM team, IDT is based in the USA and our lab sessions do not coincide with business hours. The G Block saga continued for a couple of weeks with new GC rich regions that affected synthesis identified with each modification made.

The team received the best news of the iGEM experience so far. Our G Blocks finally had no GC rich regions that inhibited synthesis. With the G Blocks arriving next prepared for the assembly and took the opportunity to spend more time planning our two aspects of human practice.

The efficiency of the competent cells produced was determined by transforming with the fluorescent constructs being used for the open day activities.

This is a trial. I do not know if this is going to work or not!

This is a trial. I do not know if this is going to work or not!

This is a trial. I do not know if this is going to work or not!