Week 10: 19^th August - 25^th August

2^nd Construction

Primer hybridation

After sequencing analysis we observed that our Golden Gate results were not significant for the 2^nd construction.
However, our sequences of natural promoter give us good results. Then we supposed that the problem of our Golden Gate come from the hybridation of our synthetic promoters.
We made new hybridations of:

Fur Binding Site 1 to 15 (5 µM)
Andersen promoter J23100 (5 µM)
RBS 0034 (5 µM)

Golden Gate

A new Golden Gate of our 2^nd construction are made with:

Fur Binding Site 1, 2 or 3 + sfGFP
Fur Binding Site 1, 2 or 3 + LacI-LVA

5 µL of each Golden Gate product are transformed into Top10 competent cells. To controlled the quality of the transformation, a negative controle (transformation procedure without plasmid) and a positive controle (transformation procedure with plasmid 1A3) are made.

Sequencing preparation

Preculture with LB medium (with carbenicillin) have been launched for Top10 transformed with our 2^nd construction, then minipreps have been realized using the miniprep kit from Machrey Nagel.

Name	Clone	Concentration	260/280	260/230	Sequencing
Fur Binding Site n°1 (with sfGFP)	1	20.2 ng/µL	2.07	1.29	Bad
Fur Binding Site n°1 (with LacI-LVA)	1	28.5 ng/µL	2.06	1.65	-
	2	25.5 ng/µL	2.01	1.84	No sequencing
	3	45.6 ng/µL	1.91	1.90	Bad
	4	54.7 ng/µL	1.83	1.27	Bad
Fur Binding Site n°2 (with sfGFP)	1	29.4 ng/µL	2.01	1.46	Bad
	2	25.5 ng/µL	2.07	2.00	Bad
	3	15.0 ng/µL	2.21	1.72	No sequencing
	4	47.1 ng/µL	1.84	1.53	-
Fur Binding Site n°2 (with LacI-LVA)	1	25.4 ng/µL	1.72	0.60	-
	2	27.7 ng/µL	1.93	1.67	No sequencing
	3	41.8 ng/µL	1.87	1.69	Bad
	4	31.1 ng/µL	1.82	1.26	Bad
Fur Binding Site n°3 (with sfGFP)	1	37.8 ng/µL	1.85	1.40	Bad
	2	78.3 ng/µL	1.87	1.85	Bad
	3	53.4 ng/µL	1.72	0.98	Bad
	4	23.3 ng/µL	2.06	2.53	No sequencing
Fur Binding Site n°3 (with LacI-LVA)	2	43.8 ng/µL	1.78	1.21	Bad
Fur Binding Site n°3 (with LacI-LVA)	4	28.2 ng/µL	2.15	2.31	-

3^rd construction

Sequencing preparation

Preculture with LB medium (with carbenicillin) have been launched for Top10 transformed with our plasmid which contains the sfGFP under the control of Lac O promoter. After one night of culture minipreps of 3 different clones have been realized using the miniprep kit from Machrey Nagel.

Name	Clone	Concentration	260/280	260/230	Sequencing
Enterobactin construction (Lac O with EntA, EntD, EntF)	1	62.7 ng/µL	2.02	0.84	-
	2	86.8 ng/µL	1.97	0.99	-
	3	112.2 ng/µL	1.98	1.23	-
	4	78.7 ng/µL	1.97	1.17	-
Enterobactin construction (Lac O with EntB, EntC, EntE)	1	80.1 ng/µL	1.92	1.01	-
Enterobactin construction (Lac O with EntB, EntC, EntE)	4	330.3 ng/µL	1.91	1.20	-