Roll up! Roll up! Help yourself to our plasmids!

Biomedical research is becoming an increasingly open and sharing environment; here, in The Grove Lab, we're all for this! We have recently been preparing a manuscript for the new open access platform created the Wellcome Trust and F1000 research, which will we post on in the future. We have also set up a lab page at Addgene, the online portal for the distribution of plasmids.

In time, we will use this platform to share all the plasmids we generate that may be useful to other researchers. To start with we've deposited a handful of plasmids encoding the core entry receptors for hepatitis C virus (HCV): CD81, scavenger receptor-B I, claudin-1 and occludin, you can read more about these in various review articles, like this or this. We use these plasmids as tools to investigate virus entry, however, these proteins also have important physiological functions, so these plasmids may be of use to others outside of HCV research.

These particular 'pDUAL' plasmids are lentiviral expression vectors that express both the gene of interest and green fluorescent protein from separate promoters. The genes  were PCR cloned by Lucas Walker (Research Assistant) from cDNA extracted from human hepatoma cells. For more on the use of lentiviral vectors see this excellent guide from Addgene.

Here is some example data using one of these vectors. We generated lentivirus encoding human CD81 and then transduced some CD81 KO Huh-7 cells. We then assessed their expression of GFP and CD81 using flow cytometry. What should be clear from the plot below is that the untransduced cells are resolutely negative, whereas the cells that received the lentivirus have GFP and CD81 galore! We're currently using this system to characterise some CD81 mutants, which we will also share soon enough.

So, please, help yourself to the fruits of our cloning!

 

CD81.jpg

The Signal to Noise Smackdown 2016

As scientists, we are only as good as our data.

We collect data using assays; this is a jargony term that essentially means a procedure for making reliable measurements of something. For instance, to assay changes in temperature throughout the year, you may take a reading from a thermometer every day at midday. Now, as it turns out we're not interested in the weather (lucky, considering there are no windows in our lab). We're interested in viruses and we have various assays to measure different things about them.

Measurements from a virus entry assay. Note the logarithmic scale which is going up by factors of 10. Virus 1 is hot. Virus 2 is not.     

Measurements from a virus entry assay. Note the logarithmic scale which is going up by factors of 10. Virus 1 is hot. Virus 2 is not. 

 

 

The quality of our data is determined by the quality of our assays. An important determinant of this is the signal to noise ratio of an assay; that is, how well can we distinguish the thing we want to measure (signal) from the irrelevant background (noise). The graph gives example data collected with one of our assays which measures the ability of hepatitis C virus to get into host cells. The noise here is the measurement made with the negative control. Virus 1 is giving a signal to noise ratio (SNR) of 355 i.e. its measurement is 355 times greater than the negative control. This is good. Virus 2 is giving a SNR of 7, this is not so good. Essentially, this means we are better able to make reliable measurements of virus 1 than virus 2.

Let the games begin.

Recently, we have been paying particular attention to the quality of our data by monitoring the SNR of individual experiments. In fact we've been having a competition: The Signal to Noise Smackdown 2016. For the last six weeks lab members have been posting the SNR for each of their experiments on a scoreboard. The winner was announced yesterday.

Drum roll...

Congratulations to Pat Kalemera, MRC PhD rotation student, with an SNR score of 2015

Thanks to the competition we now have a better idea of how reliable our assays are and we're now looking for ways to improve them, so we can really bring it for The Signal to Noise Smackdown 2017! 

The Winner

The Winner

The Trophy

The Trophy