• In order to identify the ratio between the high copy number the low to medium copy number plasmids, we analyzed the BBa_K201003 GFP production both on pSB1A2 and pSB3K3 (Fig. 4):

To test the ratio between the production of a high copy number plasmid (pSB1A2) and a low copy number one (pSB3K3), we assembled two circuits. The open loop GFP circuits are realized with a BBa_J23118 promoter and the standard biobrick I13504. From the Registry of Standard Biological Parts we knew that pSB1A2 is a high copy number plasmid while pSB3K3 is a low copy one, so the theoretical ratio between their copy number should be at least 10, but the highest value that we reached with the spectrofluorimeter was about 3,3.

Figure 4a - BBa_K201003 on pSB1A2

Figure 4b - BBa_K201003 on pSB3K3

PSB1A2 with a high copy number plasmid and a low copy number were transformed in DH5alfa bacterial cells according to the standard protocol.
One colony from each plate was picked up and let grow overnight in M9 medium at 37°C. One milliliter for each of the two samples was collected by O/N cultures and spinned at 8000 rpm for a minute; another milliliter was used for measuring the optical density and estimate the growth of the sample. The supernatant was harvested and the pellet resuspended. Slides were prepared for the acquisition of images of fluorescent bacteria.
Finally, images were elaborated with the fluorescence visualization software and these are the results:

High copy number plasmid (PSB1A2)

Low copy number plasmid (PSB3K3)

Box Plot of bacterium fluorescence. Max and minimum values are indicated by the horizontal bars.

BBa_J23100 vs BBa_J23118

• In order to identify the ratio between BBa_J23100 and BBa_J23118 promoters, we analyzed the BBa_K079031 and BBa_K079032 GFP production on pSB1A2:

Figure 5a - BBa_K079032 on pSB1A2

Figure 5b - BBa_K079031 on pSB1A2

To do this we transformed those constructs in bacterial cells; we picked up a colony from each plate and we inoculated it in M9 medium. After growing all night at 37°C we took a milliliter of each sample and we measured their optical density; than we prepared slides for the fluorescence bacteria images acquisition, following the same steps of the previous test. The images, acquired during some repetitions of the test, was elaborated with the fluorescence visualization software (VIFluoR) giving out those results: From the registry of standard parts we learnt that the strengths of J23100 and J23118 are respectively 2547 and 1429, so the ratio between them is about 1.78. Experimentally we have achieved the value of 1.2; for this reason we can say that this prove has gone well.

Open loop GFP circuit with promoter J23100 (2547)

Open loop GFP circuit with promoter J23118 (1429)

Box Plot of bacterium fluorescence. Max and minimum values are indicated by the horizontal bars.

Presence vs Absence of LacI O2 natural operator

• We needed to confirm that LacI natural operator O2 don't influence GFP production when LacI repressor is not present. We compare then the GFP expression level of BBa_K079032 and BBa_K201001

Figure 6a - BBa_K079032 on pSB1A2

Figure 6b - BBa_K201001 on pSB1A2

Following the steps of the previous tests we obtained those results:

Absence of O2

Presence of O2