Introduction - what is interlab?
Interlab is an international, collaborative experiment that aims to standardise the way that fluorescent measurements are made in order to allow comparison of absolute units between labs. This year is the 4th interlab and the big question is:
‘How close can the numbers be when fluorescence is measured all around the world?’
We are testing some RBS devices (BCDs) that hopefully will make gene expression more precise and reliable, but how will this compare around the world? These devices all very similar an example of which (TD1) is shown below in figure 1:
Figure 1: Test Device 1
We used the set protocol that we were given so as to ensure everyone was doing the same thing. These can be found at:
Our results are shown below:
Figure 1: A graph of our interlab data from the 2nd experiment
Please see this Excel File to view our raw and manipulated data.
We had some trouble in growing our TD1 cells from the very beginning. We ran the full experiment with few issues but when we looked at the optical densities of the cells, the TD1 cells had not grown. We tried several times to grow them up and we also did a growth analysis on several colonies of TD1 cells and we found that only 1 out 6 colonies grew overnight. We decided to do the full experiment again but unfortunately the 2nd time we still had poor growth for TD1 and as such we removed TD1 from our graph.
A strange phenomenon occurred in our TD3 cells in that they grew well in both of our experiments however the fluorescence was very low. We do not know why this occurred but we think it must be to do with the plasmid.
Here are some of our thoughts on the interlab project:
- We had some issues with the protocol in that sometimes it was ambiguous what it meant.
- For example at the beginning of the measuring cell fluorescence part it says the total volume will be 12 ml but on the calculation sheet it says 10ml
- We went with 10 ml as that seemed the most logical
- We added a step of sequencing the transformed plasmids to verify that we did indeed have the correct devices in the bacteria.
- We felt it would be quite easy to make this less labour intensive, provided you had the equipment
- Most plate readers can incubate and shake and then you could plate your bacteria and leave it in the plate reader for 6 hours and then get the data at the end of this meaning you wouldnt have to go in every 2 hours.
- You would then be able to take more frequent time points giving you more data
- It would also mean there is less human error as from the first pipetting to the last minutes pass so some may grow more than others.
As a team the interlab challenge was useful as it got us more acquainted with the plate reader that was very helpful for experiments on our parts. It also challenged us to think about protocols and the things that could go wrong or need to be taken into account in the experiments.
We hope that our data can be put together with everyone else's to produce some valuable and interesting results.