Team:NRP-UEA-Norwich/Week2

From 2012.igem.org

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As we are looking at producing a potential cancer therapeutic as part of our project we focused on looking at how that particular application could be used. Our main idea is to attach a nitric oxide-sensing promoter to an enzyme producing more nitric oxide (NO) in order to bring NO levels to a toxic threshold. We have found two key classes of NO-producing enzyme that may be applicable to our project: the first is nitric oxide synthase (NOS), most commonly from ''Nocardia'' species (known as NOSNoc) as it is a bacterial form of NOS, however so far we found little information on the intricacies of the enzyme and therefore feel it may not be the best enzyme to use; the other enzyme we have looked at is nitrite reductase (NiR), specifically copper-based NiR as it has a much cleaner reaction mechanism (producing just NO and H+) compared with other species of NiR.
As we are looking at producing a potential cancer therapeutic as part of our project we focused on looking at how that particular application could be used. Our main idea is to attach a nitric oxide-sensing promoter to an enzyme producing more nitric oxide (NO) in order to bring NO levels to a toxic threshold. We have found two key classes of NO-producing enzyme that may be applicable to our project: the first is nitric oxide synthase (NOS), most commonly from ''Nocardia'' species (known as NOSNoc) as it is a bacterial form of NOS, however so far we found little information on the intricacies of the enzyme and therefore feel it may not be the best enzyme to use; the other enzyme we have looked at is nitrite reductase (NiR), specifically copper-based NiR as it has a much cleaner reaction mechanism (producing just NO and H+) compared with other species of NiR.
 +
===Labs===
===Labs===
 +
Rebecca and Lukas went back into the labs today in order to try and transform the ''E. coli'' once again. Using the knowledge we had gained through research into why the previous transformations had been unsuccessful agar was used that had a reduced amount of chloramphenicol in order to try and grow more cells.
Rebecca and Lukas went back into the labs today in order to try and transform the ''E. coli'' once again. Using the knowledge we had gained through research into why the previous transformations had been unsuccessful agar was used that had a reduced amount of chloramphenicol in order to try and grow more cells.
 +
We were also visited by Josh Wright from Bioline who very kindly donated a large amount of plasmid isolation kits and PCR gel clean-up kits, as well as giving us a discount on Bioline products. This kind of support from Bioline is extremely important to our project and we are very thankful!
We were also visited by Josh Wright from Bioline who very kindly donated a large amount of plasmid isolation kits and PCR gel clean-up kits, as well as giving us a discount on Bioline products. This kind of support from Bioline is extremely important to our project and we are very thankful!
 +
Following Josh's visit Lukas, Russell and Khadija decided to try out the plasmid isolation kits for the first time as they tried to extract the chloramphenicol-resistance plasmid from some culutured ''E. coli''. The kits were extremely easy to use, however in keeping with our previous problems with growing ''E. coli'' we were unable to extract the plasmid, which was proven to us by a blank agarose gel picture.
Following Josh's visit Lukas, Russell and Khadija decided to try out the plasmid isolation kits for the first time as they tried to extract the chloramphenicol-resistance plasmid from some culutured ''E. coli''. The kits were extremely easy to use, however in keeping with our previous problems with growing ''E. coli'' we were unable to extract the plasmid, which was proven to us by a blank agarose gel picture.
 +
The protocol for all lab work can be found [https://2012.igem.org/Team:NRP-UEA-Norwich/Protocol Here]
The protocol for all lab work can be found [https://2012.igem.org/Team:NRP-UEA-Norwich/Protocol Here]

Revision as of 09:47, 25 July 2012

Header1NewGreen.png

NRP UEA iGEM 2012

 

Welcome to the NRP UEA iGEM 2012 Wiki Lab Book

Please choose the relevant link to access our diary of that week!

Week Zero | Week One | Week Two | Week Three | Week Four | Week Five | Week Six | Week Seven | Week Eight | Week Nine | Week Ten | Week Eleven | Lab Protocols | Experiments

Contents

Week 2

DESCRIPTION OF WEEK TWO TO BE UPLOADED AT A LATER DATE

Day 1

Research

While two members of the team worked in the labs on the transformation of Escherichia coli the rest of the team continued with the research into previous BioBricks and those that we felt the characterisation of would be useful to our project. As one of our proposed ideas for the project is to produce a BioBrick that can sense nitric oxide and then go on to increase nitric oxide amounts to toxic levels if appropriate as a potential cancer therapeutic we decided to look into genes for nitric oxide synthase and nitrite reductase, both enzymes that result in the production of nitric oxide. We also looked into plasmids and genes that may be used in mammalian cells as well as bacterial in order to start thinking about where our project could go once the BioBrick is synthesised.

Labs

Transformation of the three BioBricks was redone. Two controls were also set up in order to point us in the right direction with what may have gone wrong; the positive control used BRAF (BL21 pLysS) cells which contain vectors for chloramphenicol resistance; the negative control used the NEB 5-alpha cells used in the previous experiment; our aim was for the positive control to show whether the issue lies within the chloramphenicol resistance, or whether the problem lies within the competent cells themselves.

A sample of lysogeny broth (LB) as also inoculated with pre-made culture containing the BL21 pLysS cells in preparation for the next day's plasmid isolation focusing on chloraphenicol resistance.

Protocols for the experiments can be found Here

Day 2

Research

As we are looking at producing a potential cancer therapeutic as part of our project we focused on looking at how that particular application could be used. Our main idea is to attach a nitric oxide-sensing promoter to an enzyme producing more nitric oxide (NO) in order to bring NO levels to a toxic threshold. We have found two key classes of NO-producing enzyme that may be applicable to our project: the first is nitric oxide synthase (NOS), most commonly from Nocardia species (known as NOSNoc) as it is a bacterial form of NOS, however so far we found little information on the intricacies of the enzyme and therefore feel it may not be the best enzyme to use; the other enzyme we have looked at is nitrite reductase (NiR), specifically copper-based NiR as it has a much cleaner reaction mechanism (producing just NO and H+) compared with other species of NiR.


Labs

Rebecca and Lukas went back into the labs today in order to try and transform the E. coli once again. Using the knowledge we had gained through research into why the previous transformations had been unsuccessful agar was used that had a reduced amount of chloramphenicol in order to try and grow more cells.


We were also visited by Josh Wright from Bioline who very kindly donated a large amount of plasmid isolation kits and PCR gel clean-up kits, as well as giving us a discount on Bioline products. This kind of support from Bioline is extremely important to our project and we are very thankful!


Following Josh's visit Lukas, Russell and Khadija decided to try out the plasmid isolation kits for the first time as they tried to extract the chloramphenicol-resistance plasmid from some culutured E. coli. The kits were extremely easy to use, however in keeping with our previous problems with growing E. coli we were unable to extract the plasmid, which was proven to us by a blank agarose gel picture.


The protocol for all lab work can be found Here

Day 3

Day 4

Day 5

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