Team:UChicago/Plan

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Revision as of 19:58, 27 September 2013 by Nvivanco (Talk | contribs)


Construct pUB110 BioBrick

-Digest pUB110 w/ NdeI and AflII--> gel purify-->nanodrop for concentration


-Digest pUB110 linker w/ NdeI and AflII


Linker sequence: attcgtcttaaggaattcgcggccgcttctagagtactagtagcggccgctgcagcatatgtcatac

-Set up overnight ligation of digested, gel purified pUB110 and digested pUB110 linker = generates pUB110 BioBrick


-Transform into B. subtilis to amplify


-Set up O. N. cultures


-Do B. subtilis miniprep


-Digest our pUB110 BioBrick


To test pUB110 Biobrick (kanamycin resistant) construction (send for sequencing)


-Put a promoter/upstream BioBrick (in vector with chloramphenicol resistance) + RFP BioBrick from amp resistant vector => do 3 step assembly


-Do transformation in B. subtilis


-Choose transformants--> if transformants express RFP, we could conclude our pUB110 BioBrick works--> submit pUB110 BioBrick

"Construct kerA BioBrick"

-Do Gibson assembly to put together the two kerA gBlocks from IDT


-Put our kerA biobrick into an empty vector w/ amp resistance (so we can use the 3 step assembly) and transform into DH5-a


-Since promoter/upstream biobrick will be in chloramphenicol resistant vector


-And our puB110 vector will use kanamycin resistance


-So that leaves our kerA biobrick the vector that is amp resistant

Orange: prefix Green: suffix Purple: kerA signal peptide