Team:Macquarie Australia/Collaborations
COLLABORATIONS
Team Sydney_Australia (Sydney University)
We reached out to Team Sydney Australia who were also looking for a collaboration. Two team members from both teams participated in a video call to discuss projects in detail. Team Sydney Australia was working on producing psilocybin, and were having trouble expressing one of the four genes in their construct.
WHAT WE DID FOR THEM
Team Sydney_Australia provided us with a plasmid containing one of their genes, (PsiD), to see if we could get protein expression using our protocols. Carly and Jerresa visited the Sydney University campus to exchange plasmids and begin experimentation.
First, we transformed the BL21(DE3) cells with the PsiD-pET28 plasmid and plated those out on kanamycin (50 ug/mL) LB plates. After successful growth, we chose two single colonies to inoculate in two lots of 50 mL LB broths also containing 50 ug/mL kanamycin. For our negative control, we placed empty BL21(DE3) cells in a third 50 mL LB broth. The cultures were shaken at 37 °C at 200 rpm until the OD600 was at 0.5, monitoring every 20 minutes. Once OD600 0.5 was reached, the broths were separated into two, with one being induced with 1 mM IPTG, and the other left uninduced. All three cultures (induced, uninduced and control) were subsequently placed back in the shaker and left to grow overnight at 22 °C and 200 rpm.
After 18 hours, 1 mL was removed from each culture for whole cell SDS-PAGE. We harvested the cells by centrifugation at 5,000 x g for 10 minutes and removed the supernatant. The cell pellets were resuspended in 5 mL of phosphate-buffered saline, and lysed with sonication (60 % amplitude, 6 cycles, 10 seconds on, 10 seconds off). The lysates were centrifuged again for 20 minutes at 20,000 x g to separate the soluble and insoluble fractions.
The SDS-PAGE was successful, as seen in Fig. 1.
Figure 1: SDS-PAGE of Team Sydney_Australia's PsiD gene. The expected protein size is 49 kDa, and is shown in the red boxes.
WHAT THEY DID FOR US
At this stage in our project, we had just begun collecting experimental data for our riboswitch constructs [BBa_K3151011] through fluorescence assays[1]. However, we still had to work on construction of our hydrogenase constructs and had limited time. We were looking for assistance in the aggregation of data and consolidation of results regarding the expression our riboswitch constructs with varying promoters.
After receiving our plasmid samples, Team Sydney_Australia began experimentation following the protocol we provided. This protocol consisted of 5 mL of fresh LB, 5 μL of corresponding antibiotic (either kanamycin or chloramphenicol) and 50 μL of overnight culture. These were used to fill the wells of a 96 well plate, which was measured with the BMG Pherastar plate reader to measure eGFP (Ex485 nm Em520 nm) and OD (600 nm). The results of these experiments (fig. 2 & 3) corroborated our data, showing that our constructs produced reversed response depending on the promoter used.
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| Figure 2: eGFP produced by cells transformed with the Lac riboswitch constructs over 30 hours. Data provided by Team Sydney_Australia. | Figure 3: eGFP produced by cells transformed with the Tac riboswitch constructs over 30 hours. Data provided by Team Sydney_Australia. |
Team NTU-Singapore (Nanyang Technological University)
For the fourth year running, Macquarie_Australia continued communicating with NTU-Singapore. Our first video call with the team was in early July to gauge the possibility of bi-directional collaboration.
There were many ways in which we could collaborate. In previous years, Macquarie_Australia has performed protein expression analyses and in return, NTU Singapore has performed Quantitative Polymerase Chain Reaction (qPCR). Throughout our video calls and emails, we further discussed this idea with team members and advisors.
Due to strict regulations with Australian customs, we realised that shipping of mammalian cells into the country in time may be difficult. We also discussed the possibility of sending NTU-Singapore our riboswitch to test its functionality using their RNA editing tool, though our riboswitch was assembled and tested during the last month leading up to the wiki freeze.
Nonetheless, in the spirit of comradery, both teams sent their samples in an attempt to help each other regardless of time constraints. Fortunately, both teams received their respective samples.
WHAT WE DID FOR THEM
NTU-Singapore were working with at least 12 mutant constructs for their RNA base editing tool. They had not yet performed a protein expression analysis so we suggested performing an SDS-PAGE with their samples.
The protein of interest at (~150kDa) was seen in both gels (below).
Figure 4: Gel one shows a protein of ~150 kDa as a faint line, which was not seen in the control lane.
Figure 5: Gel two shows a protein (~150kDa) all the samples. This protein is not seen in the control.
The band in the SDS-PAGE gel is faint, perhaps due to degradation during international shipping. We sonicated the DNA after our first attempt because our lanes were overloaded, although we may still have too much in these final gels and further sonication may have been required. Nevertheless, we showed expression of a protein ~150 kDa, thus demonstrating expression of NTU's protein of interest via SDS-PAGE.
WHAT THEY DID FOR US
As we had already organised with Team Sydney to perform a GFP assay for fluorescence for our riboswitch, we thought it would be beneficial for our project to ask NTU-Singapore to perform a qPCR on our hydrogenase in order to confirm the correct number of genes are present in our sample.
We asked NTU to transform our hydrogenase into E. coli using our Lac R ON and Lac R OFF samples. An overnight culture was grown and liquid cultures were prepared once cultures reached OD 0.5, and subsequently induced with IPTG. We then asked the cells to sampled for RNA isolation at time points 0 hours, 4 hours, and 24 hours. Following this, the qPCR would be performed.
Team UNSW_Australia (University of New South Wales)
For the second year running, Macquarie_Australia was invited to present and attend the UNSW Synthetic Biology Symposium. iGEM teams across Sydney were invited to present a 5 minute overview of our projects in preparation for the Giant Jamboree. We would like to extend our gratitude to Team UNSW Australia for inviting us to hear about the experiences of the guest panelists and the opportunity to socialize with other iGEM teams!
Team NU_Kazakhstan
During our search for other iGEM teams working with hydrogen producing microbes, we decided to reach out to Team NU_Kazakhstan who were working on hydrogen gas production in Synechoccoccus elongatus. In late August, Jerresa and Carly participated in a video call with Team NU_Kazakhstan where we discussed our projects.
The idea was suggested to use our hydrogen biosensor to detect the presence of hydrogen gas produced by Kazakhstan’s cyanobacteria, as this would be a really interesting way of confirming both teams’ system functionality. We decided that this may not be a feasible option as we were still constructing our biosensor in E. coli and Team NU_Kazakhstan was having difficulty assembling their plasmid in cyanobacteria. There were also compatibility issues as E. coli and S. elongatus are bacteria and cyanobacteria respectively. Another potential obstacle would be that the hydrogen produced by S. elongatus in aqueous form may not be detectable with our biosensor, which may only be able to detect hydrogen gas. Although a collaboration did not progress, we would like to thank Team NU_Kazakhstan for their time and knowledge.
Team UAAAN (Universidad Autónoma Agraria Antonio Narro)
In early September, Team UAAAN reached out to us for collaboration as they were working with the 2017 Macquarie Australia iGEM gene cluster. Both teams participated in a video call to discuss our projects and possible ideas. An idea was a mentorship since this was the first time Team UAAAN was competing in iGEM and Team Macquarie_Australia’s 10th year competing. Another collaboration discussed was the characterisation of the 2017 Macquarie Australia iGEM gene cluster because they were using it without one of the four genes, and the subsequent detection of hydrogen gas using our biosensor. Due to the logistical challenges in sending parts between Mexico and Australia, we decided this collaboration may not be practical for the time limits until the wiki freeze. We wish team UAAAN the best of luck for their first time competing in iGEM!
References
- Dell EJ. Bottom reading of cell-based assays: direct optic approach enhances fluorescent protein and other microplate analyses. Genetic Engineering & Biotechnology News. 2012 May 1;32(9):22-3.
