Week 9: 12^th August - 18^th August

TECAN

Medium preparation

For the Tecan analysis we must use M9 medium and not a classical LB medium because turbidity and fluorescence of our sample are measured. Then, in order to obtain good results, the medium must not emit a side signal, that is why we use the M9 medium.

Composition for 50 mL of:

Reagent	M9 medium (without iron)	M9 medium (with iron)
M9 salt (5X)	10 mL
CaCl2 (1M)	5 µL
MgSO4 (1M)	100 µL
Glycerol (50%)	800 µL
Thiamine	5 µL
NaOH (pH 7.4)	12.5 µL
H2O	40 mL	39 mL
FeSO4 (10mM)	-	50 µL
Casamino acids (0.2%)	-	1 mL

Once the mixture is prepared, the medium must be filtered to be sterilised using 0.22 µm filter.

TECAN analysis

Using the TECAN analysis, we are measuring the capacity of repression of the natural binding site that we extract from the E.coli's genome.

Preculture with M9 medium (with carbenicillin) have been launched for BL21 transformed with our 1^st construction:

• Natural Fur Binding Site of Fec A + sfGFP (clone 1, 2, 3)
• Natural Fur Binding Site of Fep A + sfGFP (clone 1, 2, 3)
• Natural Fur Binding Site of Ace B + sfGFP (clone 1, 2, 3)

Note: Preculture have been made in M9 medium with iron in oder to inhibit the expression of sfGFP.

After one night of culture, time the precultures have been refreshed by diluting them 200 times in M9 medium (with iron and carbenicillin).

After 8 hours of culture, the 96 wells plate has been prepare (see following scheme).

AJOUTER DETAIL DES CYCLES

1^st construction

Sequencing preparation

Preculture with LB medium (with carbenicillin) have been launched for Top10 transformed with our 1^st construction, then minipreps have been realized using the miniprep kit from Machrey Nagel.

Name	Clone	Concentration	260/280	260/230	Sequencing
AceB Fur Binding Site (with LacI-LVA)	1	66.4 ng/µL	1.89	1.92	Good
	2	53.8 ng/µL	1.85	1.61	Good
	3	64.9 ng/µL	1.89	1.87	Bad
	4	71.8 ng/µL	1.84	1.66	Good
ybiL Fur Binding Site (with LacI-LVA)	1	49.1 ng/µL	1.94	1.80	Good
	2	58.6 ng/µL	1.99	2.32	Good
	3	41.2 ng/µL	1.97	2.44	Good
	4	31.8 ng/µL	1.93	2.16	Good
yncE Fur Binding Site (with LacI-LVA)	1	43.7 ng/µL	1.95	2.17	Good
	2	45.6 ng/µL	1.97	2.22	Good
	3	34.8 ng/µL	2.00	2.41	Good
	4	42.5 ng/µL	2.00	2.28	Good
Fes Fur Binding Site (with LacI-LVA)	1	39.4 ng/µL	1.89	1.82	Good
	2	36.6 ng/µL	-	-	Good
	4	35.3 ng/µL	1.81	1.13	Good
FepA Fur Binding Site (with LacI-LVA)	2	50.7 ng/µL	1.90	1.61	Good
	4	57.4 ng/µL	1.92	1.88	Good

2^nd construction

Bacterial isolation

4 clones of a Golden Gate plate are isolated for each construction. It allows us to save the clone on a plate.

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	50.7 ng/µL	1.62	0.68	Bad
	2	21.1 ng/µL	1.69	0.44	No sequencing
	3	21.6 ng/µL	1.20	0.13	No sequencing
	4	70.8 ng/µL	1.66	0.72	Bad
Fur Binding Site n°2 (with sfGFP)	1	39.1 ng/µL	1.23	0.40	No sequencing
	2	25.1 ng/µL	1.37	0.29	No sequencing
	3	61.1 ng/µL	1.57	0.71	Bad
	4	54.7 ng/µL	1.62	0.68	Bad
Fur Binding Site n°3 (with sfGFP)	2	38.4 ng/µL	1.84	0.78	No sequencing
	3	114.6 ng/µL	1.66	0.78	Bad
	4	39.2 ng/µL	1.87	1.21	No sequencing
Fur Binding Site n°5 (with sfGFP)	1	77.7 ng/µL	1.76	0.99	Bad
	3	78.4 ng/µL	1.61	0.81	Bad
	4	72.7 ng/µL	1.85	4.23	Bad
Fur Binding Site n°6 (with sfGFP)	1	70.4 ng/µL	1.76	2.16	Bad
	2	91.5 ng/µL	1.55	1.21	Bad
	3	65.4 ng/µL	1.70	1.52	Bad
	4	85.6 ng/µL	1.72	1.65	Bad
Fur Binding Site n°7 (with sfGFP)	1	53.5 ng/µL	1.76	1.93	Bad
	2	77.6 ng/µL	1.69	1.22	Bad
	3	72.3 ng/µL	1.68	1.22	Bad
Fur Binding Site n°8 (with sfGFP)	1	73.9 ng/µL	1.58	0.84	No sequencing
	2	51.3 ng/µL	1.75	1.10	Bad
	3	45.9 ng/µL	1.85	1.43	Bad
	4	46.1 ng/µL	1.88	1.61	Bad
Fur Binding Site n°9 (with sfGFP)	1	108.3 ng/µL	1.78	1.37	Bad
	2	67.0 ng/µL	1.88	1.61	Bad
	3	103.5 ng/µL	1.87	1.79	Bad
	4	28.2 ng/µL	2.06	1.76	No sequencing
Fur Binding Site n°10 (with sfGFP)	1	29.5 ng/µL	1.95	1.22	No sequencing
	2	70.0 ng/µL	1.90	1.67	Bad
	3	46.0 ng/µL	1.89	1.62	Bad
	4	55.8 ng/µL	1.88	1.44	Bad
Fur Binding Site n°11 (with sfGFP)	1	55.3 ng/µL	1.85	1.49	Bad
	2	87.1 ng/µL	1.81	1.37	Bad
	3	57.7 ng/µL	1.84	1.44	Bad
	4	21.7 ng/µL	2.06	1.20	No sequencing
Fur Binding Site n°15 (with sfGFP)	1	16.2 ng/µL	2.13	1.20	No sequencing
	2	47.4 ng/µL	1.78	1.16	Bad
	4	63.2 ng/µL	1.78	1.16	Bad
Fur Binding Site n°1 (with LacI-LVA)	1	99.7 ng/µL	1.73	0.99	Bad
	2	46.9 ng/µL	1.78	0.90	Bad
	3	55.8 ng/µL	1.87	1.75	Bad
	4	35.3 ng/µL	1.84	0.99	Bad
Fur Binding Site n°2 (with LacI-LVA)	1	43.8 ng/µL	1.83	1.01	Bad
	2	47.0 ng/µL	1.77	0.92	Bad
	3	32.4 ng/µL	1.78	1.09	Bad
Fur Binding Site n°3 (with LacI-LVA)	1	57.8 ng/µL	1.84	1.45	Bad
	2	51.3 ng/µL	1.79	1.04	Bad
	3	41.6 ng/µL	1.88	1.75	Bad
	4	112.6 ng/µL	1.86	1.81	Bad

Note: Fur Binding Sites n°4, 12, 13 and 14 are missing.

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
Testing construction (Lac O with sfGFP)	1	25.7 ng/µL	2.02	2.29	No sequencing
	2	27.0 ng/µL	2.09	1.78	No sequencing
	3	79.7 ng/µL	1.86	1.60	Good

Golden Gate

A new Golden Gate of our 3^rd construction as been made for the two plasmids containing:

1. Enterobactin biosynthesis genes (Ent A, D and F)
2. Enterobactin biosynthesis genes (Ent B, C and E)

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.

Unfortunatly, the Golden Gate reactions did not work.

Others

Miniprep of Plasmid 1A3 with RFP designed for Golden Gates

Top 10 transformed with the plasmid 1A3 designed for Golden Gate have been cultivated into 10 mL of LB medium (with carbenicillin). After an overnight culture, plasmid has been minipreped using the Machrey Nagel kit.

Sample	Concentration	260/280	260/230
1	265 ng/µL	1.84	1.78
2	237.8 ng/µL	1.82	1.67
3	240.8 ng/µL	1.84	1.81

The plasmid has been diluted as a working solution with a concenration of 80 ng/µL. Then the plasmid has been aliquoted in 50 µL into 9 different tubes.

Competent cells

Top10 competent cells had been made last week and we tested them on different antibiotics (Carbenicillin, Kanamycin, Chloramphenicol). After incubation at 37°C, overnight, no contaminant had been observed.

Site developped by Gabriel Guillocheau