• 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 (Fig. 2):

Figure 2a - BBa_K079032 on pSB1A2

Figure 2b - BBa_K079031 on pSB1A2

Dh5alpha cells transformed with BBa_K079032 and BBa_K079031 were inoculated in M9 medium O/N. The day after, samples of bacterial cells in the stationary phase were collected and slide prepared for image acquisition with the optical microscope. Images were then analyzed with the VIFluoR software to analyse bacterial fluorescence. Mean fluorescence per bacterium was 51.3± 8.3 a.u. for BBa_K079032 and 43.7±10.4 a.u. for BBa_K079031. Fluorescence ratio BBa_K079032/ BBa_K079031 was 1.20±0.4 (Table 1).

Table 1 - Promoter fluorescence ratio after microscope analysis

The same sample were collected for fluorescence analysis with the Tecan M200 fluorimeter (Table 2) and the fluorescence ratio was confirmed:

Table 2 - Promoter fluorescence ratio after fluorimeter analysis

Dilutions from the O/N grown cultures were then obtained (OD = 0.1) and cell let to grow a 37 °C in a Tecan spectrofluorimeter. Both optical density (OD; Fig. 3) and fluorescence level (Fig. 4) were analized during 12 h. Fluorescence/OD ratio is shown over time in Fig. 5.

Fig.3 - Growth curve

Fig.4 - Fluorescence

Fig.5 - Fluorescence curve over OD

At the equilibrium once again fluorescence/OD BBa_K079032/ BBa_K079031 ratio was about 1.20 (Fig. 5). A relevant experimental result is the roughly 30fold increase in the fluorescence signal from the single bacterial cell occurring during the time course. A possible explanation of this observation could rely on the required activation of the major s subunit of RNA polymerase for transcription of most of the genes expressed in the exponential growth phase (Jishage M, Ishihama A. Proc Natl Acad Sci USA 1998; 95: 4953–8. See reference section). Too low fluorescence per cell at the beginning of the monitoring, possibly too close to the lower threshold of the fluorimeter, may also explain why BBa_K079032/ BBa_K079031 ratio was clearly apparent only after 8 hrs in culture.

Open loop GFP circuit with promoter J23100 (2547)

Open loop GFP circuit with promoter J23118 (1429)

pSB1A2 vs pSB3K3

• 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):

Figure 4a - BBa_K201003 on pSB1A2

Figure 4b - BBa_K201003 on pSB3K3

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. 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.

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