Difference between revisions of "Team:Dundee Schools/Results"

Revision as of 14:32, 12 August 2016

Background Information:

The hfq is a RNA binding protein and it is specific to vibrio cholerae and shigella which means it targets these bacteria.

osmY is a protein often secreted by E. coli. This means that we can attach a specific sRNA sequence which will be formed by a plasmid in the E. coli. The osmY will then escort the sRNA out of the cell. However, the sRNA cannot bind to the osmY therefore we made an osmY – hfq fusion so it can bind to the sRNA.

sRNA is made by the expression system, Ec-sRNA E. coli and Sma-sRNA S.marcescens. Once the sRNA is outside of the E. coli we want the vibrio cholerae and shigella to take up the sRNA so that it can block a vital DNA sequence which stops translation of a protein which makes up the tail of the vibrio cholera. The protein is called fliC.

To test the ability of the sRNA we plan to run E. coli cells that have been inhibited by the sRNA on protein gel. Normally when E. coli is run on a protein gel a large band would be found at 37 kBa which would represent the huge amounts of fliC which is used to make up the tail of the E. coli. However once the translation of the fliC protein have been inhibited by the sRNA a really small band of different proteins would be found at 37 kBa, and no fliC should be found. We can also check the two samples and compare them under a microscope to see if the inhibited sample is swimming, since it has no tail it should be unable to move. Another method of testing the sRNA’s efficiency will be to plate the inhibited cells along with normal cells as a control, once plated the normal cells will be able to grow outwards as they have the ability to move due to their tail, however the inhibited cells will stay close to where they were put on as they can’t move without their tails.

The first experiment performed to characterise our system is a western blot. The western blot will separate and let us identify proteins. For the western blot we are using our system, responsible for producing the osmY – hfq fusion, which was left in an overnight culture before. Both the super-native and the cells were loaded as a sample to see if the osmY – hfq fusion was indeed being secreted from the E. coli. However to be able to see the fusion on a gel we need to add a HA tag to it.

Since osmY (BBa_K892008) has already been submitted as a BioBrick to the registry before, we plan to improve it to qualify for the gold medal award. We have added a sequence which allows the expression of a HA tag to the osmY BioBrick, which in turn allows antibodies to be able to attach itself to it and provide a good method for the detection and purification of tagged target proteins.