PSB1A7 Cloning Solution
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===Solution to pSB1A7 cloning problem: Pancake stacks without B0015=== | ===Solution to pSB1A7 cloning problem: Pancake stacks without B0015=== | ||
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| + | Our new cloning vector pSB1A7 insulates our parts from read-through transcription, but this vector does not accept parts that carry the B0015 double terminator. Our flipping device was originally designed with a B0015 near the end. Several attempts to clone parts carrying B0015 into pSB1A7 failed. This problem prompted a redesign of our system. | ||
[[Image:Pancakes_noTT.gif|600px]] | [[Image:Pancakes_noTT.gif|600px]] | ||
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| + | As shown above, we redesigned our two-pancake stack to exclude the double terminator at the end. So far, two permutations of this newly designed stack [(1,2) and (1,-2)]have been successfully cloned into pSB1A7. We've also eliminated the RE to "slown down" Hin invertase function. Construct #2 carries AraC (pBad repressor) and Hin on a second plasmid marked by kanamycin resistance. The two plasmids will be co-transformed into E. coli, and cells carrying both will be selected for using Amp + Kan media. | ||
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| + | Tests are currently under way to determine if the AraC/ Hin generator on plasmid #2 can | ||
| + | 1. '''Suppress pBad-driven transcription''' - adding arabinose C should induce transcription from pBad for pancakes that are in the proper orientation | ||
| + | 2. '''Induce inversion''' - this will be assayed by tet resistance in the presence of arabinose and NheI restriction digest | ||
Revision as of 23:04, 30 October 2006
Solution to pSB1A7 cloning problem: Pancake stacks without B0015
Our new cloning vector pSB1A7 insulates our parts from read-through transcription, but this vector does not accept parts that carry the B0015 double terminator. Our flipping device was originally designed with a B0015 near the end. Several attempts to clone parts carrying B0015 into pSB1A7 failed. This problem prompted a redesign of our system.
As shown above, we redesigned our two-pancake stack to exclude the double terminator at the end. So far, two permutations of this newly designed stack [(1,2) and (1,-2)]have been successfully cloned into pSB1A7. We've also eliminated the RE to "slown down" Hin invertase function. Construct #2 carries AraC (pBad repressor) and Hin on a second plasmid marked by kanamycin resistance. The two plasmids will be co-transformed into E. coli, and cells carrying both will be selected for using Amp + Kan media.
Tests are currently under way to determine if the AraC/ Hin generator on plasmid #2 can 1. Suppress pBad-driven transcription - adding arabinose C should induce transcription from pBad for pancakes that are in the proper orientation 2. Induce inversion - this will be assayed by tet resistance in the presence of arabinose and NheI restriction digest
