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Team:UChicago/Plan
From 2013.igem.org
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| - | == ''' | + | == '''Summer Plan''' == |
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| + | == Construct pUB110 BioBrick == | ||
-Digest pUB110 w/ NdeI and AflII--> gel purify-->nanodrop for concentration | -Digest pUB110 w/ NdeI and AflII--> gel purify-->nanodrop for concentration | ||
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-Choose transformants--> if transformants express RFP, we could conclude our pUB110 BioBrick works--> submit pUB110 BioBrick | -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 | -Do Gibson assembly to put together the two kerA gBlocks from IDT | ||
Revision as of 20:00, 27 September 2013
Summer Plan
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
