Team:Exeter/Interlab
Interlab Study
Chapter 1: New Beginnings
Once upon a time, there was an iGEM team from the University of Exeter. Determined to contribute to the field of synthetic biology, we decided to participate in the Second International Interlab Measurement Study. While determining fluorescence levels for the three Interlab devices, we hoped to gain some experience in the lab before starting our iGEM project, Ribonostics. We aimed to build and measure the fluorescence of three BioBrick devices:
- J23101 + I13504 (D1)
- J23106 + I13504 (D2)
- J23117 + I13504 (D3)
In the first week of our iGEM induction, we started out by hydrating DNA from the kit, after which we followed the standard protocols for BioBrick assembly. The first step was transforming our BioBrick DNA into competent DH5α cells (New England Biolabs), and growing them in overnight cultures. After transforming, we performed our first miniprep to obtain the DNA needed for further experiments, and checked the DNA concentration using Qubit. We then used values obtained from this to calculate the volumes of all of components of the digestion step. After the digestion, we ran a part of each sample on a gel to see if the insert and vector were the correct size. The ligation was the last step; after this, we plated our samples and incubated them overnight. We hoped that the next morning, there would be glowing green colonies on our plates. Protocols of these experiments can be found on these pages of the lab book: [transformation, miniprep, Qubit, digestion, ligation]
The next day, we came into the lab, hoping to see colonies so green they would be blinding. After viewing the plates under blue light to visualise the green fluorescent protein (GFP) in our constructs, we understood that the statement ‘biology sometimes doesn’t work’ is entirely accurate. There were no glowing colonies on our plates. However, this moment of truth inspired us to diagnose what was wrong with our devices, and give the Interlab Study another try in the following week.
Our lab induction and Interlab Study were a good chance to find out where the equipment and resources in the lab were, as well as to meet some of the researchers working there – many of them helped at various stages of our project. Those amongst us with little or no lab experience learned basic techniques, such as pouring agar and making LB broth, but also made an effort to understand the biological concepts behind the Interlab Study. One of our physicists, Todd, said that in the first week, he learned the skills necessary for any budding biologist – pipetting, making, and pouring agar. ‘I thought it was very useful. It was great.’ – a direct quote from Todd. Equipped with these skills, we set out to complete the Interlab Study accurately, efficiently, and most importantly – successfully.
Chapter 2: Under Pressure
After a weekend of thought, we came into the lab, determined to get glowing colonies by the end of the week. The answer to why our Interlab Study failed the previous week was simple: we had been trying to put the wrong BioBricks into the pSB1C3 plasmids – they did not join, because they could not join.
We looked at our second attempt at the Interlab Study with optimism – we had some practice in the lab, and we understood more clearly what it was we were actually trying to do. This time, we made and used TOP10 competent cells. Again, we followed the same flowchart of experiments, using the correct BioBricks (J23101, J23106, J23117).
This time, only three or four of us worked in the lab at any one time. During our first week in the lab, we learned that having more people in the lab does not necessarily mean we are more productive. During the second try at the Interlab Study, we also made sure we had all the necessary controls – our competent TOP10 cells alone, the empty pSB1C3 backbone, a working GFP plasmid and a negative control of the TetR plasmid. After a few setbacks (such as forgetting to put LB broth during the incubation stage of a bacterial transformation), we arrived at yet another overnight incubation, and again, we asked ourselves the burning question – would we see glowing colonies?
Chapter 3: We Fight On
By now, you may have guessed that our team is not the luckiest when it comes to glowing. If so, you guessed right – we did not have any glowing colonies.
We were baffled, puzzled, surprised – but not broken. Once again, we marched into the lab, determined to see fluorescent colonies. We double (and triple) checked that we were using the correct BioBricks, and enzymes during the digestion step. We also thought carefully about what we could change in order to get our Interlab Study to work. During our third attempt, we followed a protocol for a traditional digestion instead of the digestion protocol we had used before. We also altered the transformation protocol slightly. After running our digest on the gel, we performed a gel extraction – this way we would be sure that we were using vector and insert fragments of the correct sizes. Amongst the things we added and changed were also: control plates of just our competent cells, and using 1 µl of freshT4 ligase instead of 0.5 µl. Again, we followed the Interlab flowchart of procedures. Armed with our transformed Interlab samples and plenty of controls, we saw no reason for our colonies not to glow the next morning.