Part:BBa_I13458:Experience

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An Attempt to Use Uncoupled AraC to Regulate the Uncoupled pBad Promoter

Methods: We have attempted to use AraC I13458 to control expression from the uncoupled pBad promoter BBa_I13453 in a two-plasmid system (Davidson and Missouri Western iGEM 2006). AraC I13458 contains the Cap protein, AraC protein and polymerase binding sites (PC promoter) upstream of the AraC coding region (see Lee et al., 1981 for sequences). AraC I13458 in vector pSB3K3 and hixC-pBad-hixC-RBS-TetF-hixC (BBa_J44010) in vector pSB1A7 were co-transformed (equimolar amounts, 50 ng total) into JM109 cells. Cells containing both plasmids were selected for in liquid and on solid media containing ampicillin (100 ug/mL) and kanamycin (20 ug/mL). To assess pBad activity (tetracycline resistance), cells were streaked onto amp+kan plates +tetracycline (50 ug/mL). L-arabinose was added at a concentration of 0.5%.

Results: In its native form in the arabinose operon, the araC promoter (PC) lies adjacent to pBad; the two promoters point away from eachother. In the absence of L-arabinose, AraC protein binding facilitates a DNA loop structure that inactivates pBad (Griffiths et al., 2000). In our system we observe that pBad is active (cells are tetracycline resistant) in the absence of L-arabinose when araC and pBad are located on separate plasmids.

In the native operon, pBad becomes activated in the presence of L-arabinose. The loop structure is disrupted and AraC protein bound to L-arabinose becomes an activator (Griffiths et al., 2000). In our system, we observed that the presence of L-arabinose caused the co-transformed cells grow poorly on tetracycline, suggesting that pBad becomes inactivated (loss of tetracycline resistance) in the presence of L-arabinose.

Conclusions: It appears that AraC and pBad behave differently when they are uncoupled. In the absence of L-arabinose, pBad is silenced in the native operon while it is active in the uncoupled system. In the presence of L-arabinose, pBad is activated in the native system and silenced in the uncoupled system.

References:

• Lee, N.L., Gielow, W.O., Wallace, R.G. (1981) Mechanism of araC autoregulation and the domains of two overlapping promoters, P_C and P_BAD, in the L-arabinose regulatory region of Escherichia coli. PNAS.
• Griffiths, A.J.F., Miller, J.H., Suzuki, D.T., Lewontin, R.C., Gelbart, W.M. (2000) Regulation of Gene Transcription: the arabinose operon. An Introduction To Genetic Analysis. Chpt. 11. W.H. Freeman and Co.

--Kahaynes 21:32, 7 December 2006 (EST)

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