Team:Evry/Notebook/w9

From 2013.igem.org

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     <p>Fur Binding Site n°3
     <p>Fur Binding Site n°3
     <br/>(with LacI-LVA)</p>
     <br/>(with LacI-LVA)</p>

Revision as of 14:36, 24 August 2013

Iron coli project

Week 9: 12th August - 18th 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)
CaCl2 (1M) 5 µL
MgSO4 (1M) 100 µL
Glycerol (50%) 10 mL
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 1st 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

2nd construction

Sequencing preparation

Preculture with LB medium (with carbenicillin) have been launched for Top10 transformed with our 2nd 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.

3rd 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 3rd 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)

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.

AJOUTER LES RESULTATS DE MINIPREPS

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