Team:UCL/Overview
<!DOCTYPE html>
LABS: OVERVIEW
Superoxide Dismutase Gene therapy
CHARACTERISATION
Every great idea is turned to reality through actions. Following the design of our BioBricks, we carried out an array of experiments in order to characterise our BioBricks in experimental environments. Our project was varied and involved working with two different chassis - Pichia pastoris and Escherichia coli. We also used Streptococcus mutants and a Lentiviral vector in our experiments. See more about the protocols here.
Cellular adaptation due to changes in pH affects cellular functions which could have an influence ageing at the cellular level. Our modification of the Spy-GFP to a pH sensitive promoter was done to measure pH changes within the cell due to a change in the pH of the extracellular environment. See discussion of how the pH links with our dental devices here
A characteristic feature of cellular aging are the damages caused by reactive oxygen species (ROS) due to low intracellular concentration of antioxidants. Our response to this problem was to characterize our lycopene-mNARK promoter . We assayed how different stimulated stress conditions induced the mNARK promoter to produce lycopene and how the expression of this protein enabled cell survival in different stress conditions.
For our experiment with lubrication gel and IrrE, we wanted to show that bacteria transformed with IrrE was better adapted to live in Lubrication gel compared to wild type E.coli cells. This concept is based on the possibility of employing IrrE transformed cells to detect different pathogens (that is in personal lubricant for STIs or catheter lubricant for UTI-causing bacteria) and produce a simple visible colour change.
Furthermore, we tested the feasibility of employing SOD3 for gene therapy using lentil virus vector. Mammalian cells, HeLa, were transfected with lentil virus consisting of SOD 3 gene and the reported protein, GFP. GFP titration, where we changed the volume of lentil virus and measured the amount of positive cells transfected with the virus were carried out to test our system.
In summary, we have characterized:
The Spy-GFP, showing that SPY GFP could be used as a pH sensitive promoter due to its ability to measuere pH changes in experimental conditions.
Our lssycopene-mNARK, showing that cells which expressed lycopene could still survive and grow within certain stimulated stressed environments.
The IrrE, showing that IrrE protein enabled E.Coli to grow better in lubrication gel.
