Part:BBa_K2082112

Modified Pbad

This part is a modified version of BBa_K808000. The promoter sequence was changes according to the synthetic P_BAD to decreases basal expression level.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 1144
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 979
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI site found at 961

We started by choosing the araC-P_BAD regulatory unit from BBa_K808000. This promoter is known for low basal expression and is therfore the perfect regulatory unit for the expression of potentially toxic mutator genes. Furthermore we wanted to modify this promoter to yield an even lower basal activity. Therefore, we screened the synthetic P_BAD from iGEM DTU-Denmark 2013 and chose the promoter from Col.13. Advantages of this promoter are the very low basal activity while still maintaining a high activity when induced.

Figure 1: Alignment between promoter sequence from BBa_K808000 (wt-P_BAD ) and the modified P_BAD sequence.

In addition to the low basal activity, the P_BAD has the advantage of tuneable experssion strength, that is controlled by addition of different amounts of arabinose. We estimated that controllable amounts of mutagenic proteins would also result in different amount of mutagenesis, thus enabling a tuneable mutagenesis.
In addition to the titrable promoter activity by adding arabinose P_BAD can be repressed by adding glucose. Glucose decreases intracellular 3',5'-cyclic AMP concentration, thereby lowering expression of P_BAD promoter (Miyada et al. 1984).

We characterized our modified P_BAD BBa_K2082112 by adding the RFP expression system BBa_K516032 downstream and measurement of RFP after different amounts of time and with various amounts of arabinose added. The measurement was performed in the Top10 strain, which is deficient for arabinose metabolism (araD139 Δ(ara-leu)7697).

Figure 2: Measured expression of BBa_K2082112 in comparison to BBa_K808000. RFP expression via BBa_K516032 was used for promoter characterization, and the promoter was induced by addition of arabinose. After 300 min RFP fluorescence was normalized on OD₆₀₀.

No difference in leakiness could be observed. The addition of 20 mM glucose represses the promoter slightly and decreases promoter strength by about 65 % to 52 %, compared to no addition. Addition of arabinose acitivates the promoter, in which increased amounts of inductor leads to increased promoter activity. In total is the modified P_BAD BBa_K2082112 a bit weaker than BBa_K808000. The main aim in decreasing promoter basal activity was not proven. Perhaps the used method has to be optimized further for detection the already small basal activity of BBa_K808000 and compare it with our modified promoter.

Miyada, C. G.; Stoltzfus, L.; Wilcox, G. (1984): Regulation of the araC gene of Escherichia coli: catabolite repression, autoregulation, and effect on araBAD expression. In: Proceedings of the National Academy of Sciences of the United States of America 81 (13), S. 4120–4124.