840:153g:Projects/project5

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Team Members

  • Alex Conyers
  • Jessica Rigby

Project Description

  • After doing a little bit of research, we have decided to manipulat the already existing Nar promoter from E. coli to be able to work with a RFP (Red Flourecent Protein) to indicate when the bacteria is under anaerobic conditions, and intensified while in the presence of nitrate.
  • Our main goal is to get the promoter to work under anaerobic conditions only. The bacteria will hopefully appear red without the presence of Oxygen, and appear normal in the presence of Oxygen.
  • Our first steps will include Isolating the RFP part[1] from the 2008 spring registry and transform some E. coli cells with the DNA. We will need to make sure the cells have the DNA in them by plating them on selective media.
  • Once we verify that the cells contain our part, we will extract the DNA from the bacteria, and Isolate our selected part by running it through a gel.
  • After we're certain we have the part from the registry, we will be ordering some short DNA sequences to be made for our project. These sequences were found from a few articles about our promotor, and are said to be the sections that are needed from a promoter that contains many other "unnessesary" bases. We are testing out if these sequences are in fact the only nessesary parts, or if there may be some other hidden sequence that makes this promoter work properly. These articles can be found at [2] and [3] The first article is the one we used for the precise sequences being used. (sequences can be found on page 19 of the article)
  • Our sequences are as follows
        * 5' AGATCTTTGATNNNNATCAAGGATCC 3' - Fnr
        * 5' AGATCTTTGATCCTGCTACANNNNNTACCCCGATCGGGGTAGGATCC 3' -Wild
        * 5' AGATCTTACYNMTGGATCC 3' -NarL
        * 5' AGATCTTTGATNNNNATCAANNNNNTTGATNNNNATCAAGGATCC 3' -FnrFnr
        * 5' AGATCTTTGATNNNNATCAANNNNNTACYNMTGGATCC 3' -FnrNarL
        * 5' AGATCTTACYNMTNNNNNTTGATNNNNATCAAGGATCC 3' -NarLFnr
        * 5' AGATCTTTGATNNNNATCAATACYNMTGGATCC 3' -FnrNarL2
        * 5' AGATCTTCGATNNNNATAAAGGATCC 3' -Fnr2
  • Once we have a working part, and our DNA sequences, we will be cutting the part and one of our sequences at a time, and mixing the part with our sequence to incorporate our sequence into the plasmid part.
  • These newly recombinated plasmids will then be used to transform more bacteria.
  • These bacteria will be selected for to see if our plasmid is in them, and then tested to see if our sequence will work as a promoter.
  • we will select bacteria that has been transformed by plating them on media with ampicilin, (the plasmid is suposed to have the ampicilin resistance gene in it) and test these bacteria in an anaerobic chamber to see if our promotor will actually work, depending on which sequence was put into the plasmid.
  • Some of our sequences should code for anaerobic promotion, some nitrate promotion, and some for both.
  • After we have cells that have obtained the plasmid, we will test them to see if the promoter we have inserted into the plasmid works. This will be shown by plating these cells on media with xgal and nitrate and putting these plates in an anaerobic chamber. The cells whos promoter that is activated by anaerobic growth should turn blue in the presence of xgal. The cells who's promoter that is enhanced in the presence of nitrate should turn a darker blue. This will be due to the properties of our plasmid part selected from the registry. If none of the cells turn blue, then that particular promoter did not work.


Potential Problems

  • We might not be able to extract DNA from out part in the registry, as it is only avaliable in the Spring 2008 registry.
  • We could be overlooking critical parts of the working natural promoter that allows it to function properly.
  • We're not sure if adding this extra promoter will effect the natural functions of the cell.

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