Davidson:PLac Tet Pancake Plan

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pBad Is a Weak Promoter

In our previous design, we aniticpated that a reverse RBS-RFP reporter would distinguish the biologically equivalent (1, 2) and (-2, -1) configurations of a pBad, TetR two-pancake stack. To test this, I placed the reverse RBS-RFP reporter to the left of (-2, -1). I also placed it to the left of (-1, -2) to see if the distance between the pBad promoter and the RFP would affect expression.

Only RFPrev-RBSrev-(-1, -2) confers weak expression of RFP (faint pink cell pellet), even after 0.2 - 2.0% arabinose induction in the absence of an extragenic copy of AraC (repressor of pBad). This result suggests that JM109 cells have endogenous AraC that represses pBad and that pBad is a weak promoter that requires close proximity to its coding region. Since one of our goals is to build long multi-coding sequence pancake stacks, pBad is a poor choice for this device (but may be useful in controlling Hin expression).

pLac Tet Pancake Assembly Plan

We've observed that pLac promotes strong expression of RBS-RFP, even in the absence of an inducer (IPTG). pLac may be better suited for our pancake stack device.

450px Restriction Mapping: PvuII sites occur once in the stationary RFP reporter and once in pLac. A PvuII digest can be used to check the orientation of pLac anywhere in the stack. Since PvuII and NheI share optimal buffers (Promega), a simple double digest could be used to check the orientation of pLac and Tet relative to eachother.

Construction, Step 1: First, I will build a hix-flanked pLac promoter (pLac single pancake) with a reverse RBS-RFP to the left and RBS-TetF to the right. If pLac can read through hix, the cells will be tetracycline resistant.

450px Construction, Step 2: Next, I will cotransform the pLac single pancake construct with the Hin-LVA expression cassette and induce Hin expression with IPTG. Flipping should yeild constructs with reverse pLac promoters resulting in RFP expression. Successful flipping will...
  • indicate that Hin can invert a strong promoter (pLac).
  • produce a reverse pLac (after flipping, plasmids will be purified and transformed into cells to produce clonal plasmids containing reverse pLac)
  • determine whether reverse pLac can promote reverse RBS-RFP expression
450px Construction, Step 3: A hix site will be added to the end of the unflipped and flipped pLac constructs to produce two-pancake stacks (1,2) and (-1,2). Using the same series of steps (1 through 3) with a reverse RBS-Tet, two additional stacks [(1,-2) and (-1,-2)] could be created.

In order to create the permutations with RBS-Tet in front of pLac [i.e. (2,1)], the reverse RBS-RFP reporter would have to be omitted so that more parts could be added to the left of RBS-Tet.