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We decided to create and developed a biological circuit in which the TRANS-repressor, in absence of IPTG, can inhibit the synthesis of LacI repressor protein, warranting the production of GFP:

To do that we initially need to characterize some sub-circuits in order to obtain some information and values concerning the processes we were analyzing.

You can find here all our Wetlab Protocols

Plasmid copy number characterization

To test the ratio between the production of an 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 1429 promotor and the standard biobrick I13504.

PSB1A2 with 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 LB medium at 37°C. One milliliter for each of the two samples was collected by O/N cultures and spinned at 6000-8000 rpm for three minutes. The supernatant was harvested and the pellet resuspended. Slides were prepared for the fluorescence bacteria image acquisition.
Finally, images were elaborated with the fluorescence visualization software and these are the results:

High copy number plasmid (PSB1A2)

Low copy number plasmid (PSB3K3)

Promoter characterization

In order to estimate the difference in strength of the two promoters J23100 (2547) and J23118 (1429), we realized two circuits. Both were composed by an open loop GFP mounted on an high copy number plasmid (PSB1A2), the only different element was the promoter.

Open loop GFP circuit with promoter J23100 (2547)

Open loop GFP circuit with promoter J23118 (1429)

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 1.8. Experimentally we have achieved the value of 1.2; for this reason we can say that this prove has gone well.

GFP production in absence/presence of operator Ox

To be sure that the presence of the operator Ox doesn't significantly affect the GFP production we tested two open loop GFP circuits, one with the operator Ox and another without.

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 To do that we initially need to characterize some sub-circuits in order to obtain some information and values concerning the processes we were analyzing.
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-You can find here all our [[Wetlab Protocols]]
+You can find here all our [[Team:Bologna/WetlabProtocols|Wetlab Protocols]]
 = Plasmid copy number characterization =