PSB1A7 Cloning Solution

From 2006.igem.org

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 double terminator upstream of Hin should block read-through transcription. PC-AraC (reverse) should be constitutively expressed and does not require insulation in our system. Plasmids #1 and #2 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