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From 2013.igem.org

Contents 1 Parts 2 Notebook 3 Protocols 4 Safety 5 Experiments 6 Results

Parts

Notebook

Protocols

1. Solid and liquid culture media 2xYT

For 1 liter of liquid medium:

- 16 g of triptone - 10 g of yeast extract - 5 g NaCl - Add ddH2O (di-deionized) to 1000 mL

For 1 liter of solid media

- Same compounds as liquid medium - 3.95 grams of agar to 250 mL of liquid medium

2. Chemically competent cell preparation 1. In 5 mL of 2xYT media inoculate a clone of Escherichia coli and let it grow overnight, 37°C, 180 rpm. 2. Inoculate 2 mL of E. coli culture in 200 mL of liquid culture medium in a recipient of 2 L. Grow it at 37°C, 250 rpm, until it reaches OD590 0.3 or 0.4. 3. Divide aliquots of 50 mL in 4 conical tubes and let it in ice from 5 to 10 minutes. 4. Centrifuge for 7 minutes, 4°C, 3000 rpm (~1600 x G). 5. Purge the supernatant and resuspend each pellet obtained in a recipient with 5 mL of cold solution of CaCl2. 6. Centrifuge the cells for 5 minutes, 4°C, 2500 rpm (~1333 x G). Repeat step 5 and let the cells in ice for 30 minutes. 7. Repeat step 6, but using 1 mL of cold solution of CaCl2 to resuspend the cells. Note: In this solution, cells can stay from 12 to 24 hours. 8. Divide the cells in aliquots of 100 uL and freeze it at -80°C.

CaCl2 100 mL 500 mL - 60mM CaCl2 0.882 g 4.4106 g - 15% glicerol 15 mL 75 mL - 10 mM PIPES 0.3785 g 1.8925 g

 - Sterilize (autoclave) and store it in a room temperature
 

Note: Do not use PIPES free acid

 

3.CoCl2 solution preparation (250 mM)

- Weight 0.3246 g of CoCl2 (1 M = 129,84 g). - Add 10 mL H2O to cobalt. Homogenize mixture. - Filter it using a 0.22 um strainer.

4. DNA digestion

A) Single reaction (10 uL) - DNA 125 ng - Buffer 10X* 1.0 uL - BSA 10x 1.0 uL - Enzyme* 0.25 uL - ddH2O (di-deionized) Complete to 10 uL

• Enzymes and buffers used

Enzyme Restriction site Buffer EcoRI G↓AATC ECO SpeI A↓CTAGT Tango 1x XbaI T↓CTAGA Tango 1x PstI CTGCA↓G Orange

 

B) Cobalt promoter and reporter

DNA Buffer BSA 10x ddH2O Enzymes RCNA 5 uL 2 uL 2 uL 10 uL EcoRI + SpeI 0.5 uL YFP 3.2 uL 2 uL 2 uL 11.3 uL XbaI + PstI 0.5 uL + 1 uL Plasmid 10 uL 2 uL 2 uL 5 uL EcoRI + PstI 0.5 uL

C) TorCAD and Chloramphenicol plasmid resistance (PSB1C3)

DNA Buffer Orange 10x BSA 10x ddH2O Enzymes - PSB1C3 25 ng/uL 2 uL 2 uL 5 uL EcoRI + PstI 0.5 uL - TorCAD 10 ng/uL 2 uL 2 uL 5 uL EcoRI + PstI 0.5 uL

- 37°C for 4 hours

5. Ligation A) RCNA-YFP-PSB1A3

- Plasmid 2 uL - RCNA 2.5 uL - YFP 2.5 uL - Buffer 10x 1 uL - T4 DNA ligase 1 uL - ddH2O (di-deionized) 1 uL - Incubate it overnight at 4ºC

 

B) PSB1C3 and TorCAD (10uL) - Plasmid 2 uL - TorCAD 2.5 uL - Tampon 10x 1.0 uL - T4 DNA ligase 1 uL - ddH2O (di-deionized) 3.5 uL - Incubate it overnight at 4ºC

6. Transformation Protocol 1. Start thawing the chemically competent cells on ice. 2. Add 1 - 2 uL of DNA to the 2 mL tube. Pipet up and down a few time, gently. Make sure to keep the competent cells on ice. 3. Close the tubes and incubate the cells on ice for 30 minutes. 4. Add cells tubes by immersion in a preheated water bath at 42 °C for 60 seconds. 5. Incubate the cells on ice for 5 minutes. 6. Add 400 uL of 2xYT media (make sure that the broth does not contains antibiotics and is not contaminated) to each transformation. 7. Incubate the cells at 37°C for 1 hour while the tubes are rotating or shaking. Important: 2 hours recovery time helps in transformation efficiency, especially for plasmid backbones with antibiotic resistance other than ampicillin. 8. Label two petri dishes with 2xYT agar (AMP or CHL). Plate 20 uL and 200 uL of the transformation onto the dishes, and spread. This helps you ensure that you will be able pick out a single colony. 9. Incubate the plates at 37°C for 12-14 hours, making sure the agar side of the plate is up. If incubated for too long the antibiotics start break down and untransformed cells will begin to grow, because the resistance enzyme will be excreted by the bacteria inactivating the antibiotic outside of it. 10. Pick a single colony, make a glycerol stock, grow up a cell culture and miniprep.

 

7. Miniprep

- We used Promega and Invitrogen kits. We followed manufacturer’s indications .

8. PCR Protocol

Compound Volume Program Cilcles Repeat 1. ddH2O 8 uL - - - 2. Buffer IB 10x 1.5 uL - 10 min - 3. dNTP’s 2.5 mM 1.5 uL 94 °C 1 min 1x 4.. Primer VF2 10 uM 0.4 uL 94 °C 1 min 30x 5. Primer VR 10 uM 0.4 uL 50 °C 1.5 min 30x 6. Taq 5u / uL 0.2 72 °C 10 min 30x 7. DNA 3 uL 72 °C 10 min 1x 8. Final volume 15 uL 4° C - -

Note:  Using a thermocycling on steps 3 to 7 

9. Fluorimetric assay for RCNA-YFP activation

1. Measure OD 600 of cultures to be assessed. 2. Dilute cultures to OD 600 = 0.02, in a volume of 3 mL. 3. Prepare a solution of CoCl2 1 mM, using 6 uL of 250 mM solution ( look protocol 3) and 1494 uL of 2xYT media. 4. Make other 6 solutions using the mixture on step (3). 50 uM -> 50 uL solution + 950 uL of media 2xYT 100 uM -> 100 uL solution + 900 uL of media 2xYT 150 uM -> 150 uL solution + 850 uL of media 2xYT 200 uM -> 200 uL solution + 800 uL of media 2xYT 250 uM -> 250 uL solution + 750 uL of media 2xYT 300 uM -> 300 uL solution + 700 uL of media 2xYT 5. Add 100 uL of the cultures from step (2) in each well completing 21 wells. Separating appropriately the cultures in triplicate, add to each column a different solution from step (4). 6. Add 100 uL of media 2xYT in another wells completing 21 wells, to discard the noise from media 7. Prepare the designed wells from step (5) adding 100 uL of cobalt solution from step (3) 8. Seal the plaque 9. Read fluorescence: 514 nm (excitation) and 527 nm (emission), every 15 minutes, for 16 hours.

Note: It is not recommended read absorbance using black plaque as it causes interference on reading, despite of it is better for fluorimetric assay. Instead of it, use transparent plaques.

Safety

Experiments

Results