Minnesota/23 September 2008

Measurements

Collection of Parts:

1. Plasmid pSB3K3 was obtained from paper DNA, sample plate 1014, well number 1F, and transformed in db.1 cells (as it has ccdb toxin). It is a low copy number kanamycin resistant plasmid and was used as a backbone vector. The circular plasmid was linearized by PCR using primers which have EcoRI and PstI restriction sites. Linear plasmid thus obtained, was again digested with EcoRI and Pst I and cleaned by gel elution.

2. Plasmid pSB1A2_E0240 was obtained from paper DNA, sample plate 1001, well number 4B, transformed in TOP 10 cells. It is a high copy number Amp resistant plasmid. This plasmid was used to get part, RBS-GFP-terminator. Plasmid was digested with xbaI and PstI restriction enzymes. Part of interest (876 bp) was cleaned by gel elution.

3a. To get the reference standard promoter (BBa_J23101), paper DNA was transformed into db.1 cells and digested with Eco RI and speI enzymes. The digested fragment was cleaned by ethanol precipitation. The size of the reference standard promoter is 35 bp. (was obtained from source plate 1002, well number 10F).

3b. To get the test promoters, plasmids obtained from gene art having dual repressed promoters were digested with Eco RI and speI enzymes and cleaned by ethanol precipitation.

Ligation of parts followed by transformation:

4. A three-way ligation was done and ligase mix was transformed into TOP 10 cells. The cells were plated on kanamycin plates and incubated overnight at 370 C. Next day all the clones were transferred carefully on a new kanamycin plate (to preserve the individual clones) and inoculated separately into LB media to do plasmid isolation. Recombinant plasmids were digested with EcoRI and PstI, a ~1kb fragment fall out confirmed the clone (please see the attached gel picture). Description of the Gel Picture: From left to right, 1KB DNA molecular weight marker 1. Digested standard promoter +GFP (911 bp), 2. Uncut plasmid 3. Digested lac/lambda + GFP (~1026 bp), 4. uncut of the same, 5. Digested tet/p22mnt (~950 bp), 6. uncut of the same. 100 bp molecular weight marker.

Measurement of Promoter activity:

5. Three clones from all the constructs were grown in LB media (tried minimla media without success) over night (~16 hours) along with TOP 10 cells (control). Next day 1% inoculum was used to inoculate 5 ml fresh culture media. Cells were allowed to grow for 3 hours at 370 C, 250 RPM. After 3 hours Cells were aliquoted in 96 well plate in triplicates. Fluorescence and optical density were measured simultaneously. Fluorescence was normalized w.r.t. opical density and plotted on a bar graph (Figure.1.). Abbreviations are as follows: Top10 is the control, Stp1-3 are three clones with the standard promoter, lac1-3 are three clones with dually repressed promoter for LacI/lambdaCI (part K101001) and tet1-3 are three clones with dually repressed promoter for TetR and P22MNT (part K101000). As you can see in the figure, compared to standard promoter, dually repressed promoters are weaker, though they definitely have promoter activity.