Team:Paris/Modeling/Protocol Of Characterization

Principles of the Experiments

To evaluate quantitativly the activity of a promoter in function of its transcription factors, we need data in which the different values of the activities are correlated with various known and controlled values of the transcription factors concentrations. Therefore, we designed a generic plasmid in which the transcription factors are put under the control of previously characterized inducible promoter, and the studied promoter is put before a fluorescent reporter gene. In order to allow the study of the influence of two transcription factors over the tested pormoter, we chose to put the tested genes under two different inducible systems . One is the pBAD-AraC system. The second one is an indirect system were the gene is after the Tet inducible promoter « pTet ». The TetR gene would be expressed constitutively and at high rate thanks to a strong promoter (J23101) and its influence over the pTet promoter would be regulated by the concentration of the aTc molecule. That way the production of the tested gene can also be regulated. The J23101 and the pTet have been previously characterized. Here we show the design of two plasmids : one to test the influence of one gene and the other to test the influence of two genes over the tested promoter.

I-Plasmid for promoter characterization

I-A-For study with one Transcription Factor

I-B-For study with two Transcription Factor

II-Molecular design for Promoter Characterization Plasmid

Our aim is to make this plasmid useful not only for our project but for the whole iGEM comunity. This is why we decided to keep the Biobrick spirit as much as we could, making the plasmid compatible with the parts, so the teams using it need only the four traditional enzymes: EcoRI, XbaI, SpeI and PstI.We wanted also to make optional the introduction of a second tested gene. The strategy is then based in two plasmids. The principal plasmid contains everything needed to test the effect of one gene over the tested promoter activity. The second plasmid called « Accessory plasmid » can be introduced easily in the Principal Plasmid and contains the necessary elements to add the expression of a second gene to the system. The resulting plasmids are presented below.

II-A-Principal plasmid

II-B-Accessory plasmid

III-Promoter and Transcription Factors insertion

The strategy to introduce the tested promoter and the tested gene(s) is very simple. The only difficulty is that the order of insertion that we describe has to be respected to avoid unwanted restriction enzyme cuts:

• 1st step: Introduce the tested promoter:

-Cut Promoter with EcoRI and SpeI -Cut Principal plasmid with EcoRI and XbaI

• 2nd step: Introduce the 1st tested gene:

-Cut Gene with XbaI and SpeI -Cut Principal plasmid+Promoter with SpeI -Check the right orientation of the 1st gene by PCR with appropiated primers

If wanted…

• 3rd step: Introduce the 2nd gene into the Accessory plasmid

-Cut Gene with XbaI and SpeI -Cut Accessory Plasmid with XbaI and SpeI -Check the right orientation of the 2nd gene by PCR with appropiated primers4

• 4th step: Introduce the 2nd gene expression system into the Principal Plasmid:

-Cut Accessory Plasmid+2nd gene with PstI -Cut Principal plasmid+Promoter+1st gene with SpeI -Check the right orientation of the 2nd gene expression system by PCR with appropiated primers

IV-Resulting plasmids

IV-A-For one transcription factor effect

IV-B-For two transcription factors effect

V-Protocole for plasmid construction

We show here our plan to make these plasmids at the experimental level.

V-A-Principal Plasmid construction

We devided the Principal Plasmid in two main blocks to go faster:

V-A-1-Blocks

V-A-2-Block 1 construction

the result...

V-A-3-Block 2 construction

Biobrick assembly steps:

Restriction sites addition by PCR:

V-A-4-Two-block assembly

V-B-Accessory plasmid construction

Addition of restriction sites to B0015 by PCR:

Addition of restriction sites to pBAD-AraC by PCR:

Assembly:

Assumptions Specific to the Experiments

The previous experiment is used to find parameters regarding to our modelization. However, the experiments themselves must be described in the same way to involve those parameters in a consistent way, and actually, to be interpreted.