Difference between revisions of "Part:BBa K818600"

Revision as of 20:01, 26 September 2012

AmilGFP with sboA regulator

This is a coding construct for B. subtilis to produce AmilGFP pigment in response to rotten meat volatiles in the environment. AmilGFP is a yellow pigment designed by iGEM Uppsala 2011 and the regulator, sboA, is designed by iGEM Groningen 2012.

Usage and Biology

Expression in E. coli

SboA-AmilGFP is strongly expressed in E. coli, on plate and in liquid culture, at normal growth conditions. On plate, the yellow colour is less visible compared to the cell pellet in liquid culture.

Left: Pellet of SboA-AmilGFP in E. coli dh5a. Right: Plate with SboA connected to several pigment genes inside E. coli dh5α. B3 is SboA-AmilGFP

Expression in B. subtilis

SboA-AmilGFP was shown to be very weakly expressed in Bacillus subtilis on LB plate (faint color formation after 2 days). This is probably due to the leakiness of the promoter.

We (iGEM Groningen 2012) tested the expression of SboA-AmilGFP in B. subtilis subjected to volatiles from spoiled meat using the same setup as we used for the microarray (see our [http://2012.igem.org/Team:Groningen/Sensor| sensor page]).

First, we inoculated B. subtilisSboA-AmilGFP and B. subtilisWildtype from plate into flasks of Luria Broth: 1) Subjected to spoiled meat 2) Without meat

We grew B. subtilis containing sboA-AmilGFP device in the setup overnight (16 hours) at 37 degrees Celsius. In the picture below, you can see the result: B. subtilisSboA-AmilGFP strain that was subjected to spoiled meat had turned bright greenish yellow (even visible in liquid LB culture), while the same strain that was grown without meat only showed very faint yellow color. Both B. subtilis wildtype in this setup did not express yellow color at all.

Left:from left to right: Wildtype grown without meat, B.s.(SboA-AmilGFP) grown without meat, Wildtype grown with spoiled meat, B.s.(SboA-AmilGFP) grown with spoiled meat, two jars of spoiled meat. Right: Pelleted cells after 16 hour growth with/without spoiled meat.

To check whether the difference in color was not the result of the promoter activation by the presence of meat in general, we also compared the growth of B. subtilis SboA-AmilGFP strain subjected to fresh meat and rotten meat. We grew the strain in Luria Broth in the microarray setup for 12 hours and measured OD (600 nm), Absorbance (395 nm) and assayed the color of the cells when pelleted. In the curves below you can see the results: while grown without meat volatiles and with fresh meat volatiles, our device strain still produces yellow color. The color was produced faster and in a larger amount when the device strain was subjected to volatiles from spoiling meat.

Left: Absorption of AmilGFP (395 nm) per amount of cells (OD(600)) of Bacillus subtilis SboA-AmilGFP strain grown for 12 hours while subjected to spoiled meat, fresh meat, or no meat. Right:Visibility of yellow color of pelleted cells by eye. Assay done with 5 previously made pellets of different color intensities as a reference to ensure objectivity.

Sequence and Features