From 2013.igem.org

Protocols

Solid and liquid culture media 2xYT

For 1 liter of liquid medium:

• 16 g of tryptone
• 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.

Chemically competent cell preparation

• In 5 mL of 2xYT media inoculate a clone of Escherichia coli and let it grow overnight, 37°C, 180 rpm.
• 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.
• Divide aliquots of 50 mL in 4 conical tubes and let it in ice from 5 to 10 minutes.
• Centrifuge for 7 minutes, 4°C, 3000 rpm (~1600 x G).
• Purge the supernatant and resuspend each pellet obtained in a recipient with 5 mL of cold solution of CaCl2.
• 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.
• 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)
• Divide the cells in aliquots of 100 μL and freeze it at -80°C.

CaCl₂ solution (100 mL):

• 60 mM CaCl₂ - 0.882 g
• Glycerol 15% - 15 mL
• 10 mM PIPES - 0.3785 g (Note: Do not use PIPES free acid
• Sterilize (autoclave) and store at room temperature

CoCl₂ solution preparation (250 mM)

• Weight 0.3246 g of CoCl₂ (1 M = 129.84 g).
• Add 10 mL H2O to cobalt. Homogenize the mixture.
• Filter it using a 0.22 μm strainer.

DNA digestion

A) Single Reaction (10 μM)

  DNA ---------------------------------------- 125 ng
  Buffer 10X* -------------------------------- 1 μL
  BSA 10 μg/mL ------------------------------- 1 μL
  Enzyme* ------------------------------------ 0.25 μL
  ddH2O (di-deionized) ----------------------- complet to 10 μL

^*Enzyme and buffers used:

B) Cobalt promoter (RCNA) and reporter (YFP)

C) TorCAD and Chloramphenicol plasmid resistance (PSB1C3)

• 37ºC for 4 hours

D) TorCAD and PSB1C3-RFP

• 37ºC for 4 hours

Ligation

A) RCNA-YFP-PSB1A3 (10μL)

   Plasmid (PSB1A3) --------------------- 2 μL
   RCNA (BBa_K540001) ------------------- 2.5 μL
   YFP (BBa_E0430) ---------------------- 2.5 μL
   Buffer 10X --------------------------- 1 μL
   T4 DNA Ligase ------------------------ 1 μL
   ddH2O (di-deionized) ----------------- 1 μL

• Incubate it overnight at 4ºC.

B) PSB1C3 and TorCAD (10μL)

   Plasmid (PSB1C3) --------------------- 2 μL
   TorCAD (BBa_K1086000) ---------------- 2.5 μL
   Buffer 10X --------------------------- 1 μL
   T4 DNA Ligase ------------------------ 1 μL
   ddH2O (di-deionized) ----------------- 3.5 μL

• Incubate it overnight at 4ºC.

C) PSB1C3-RFP and TorCAD (10μL)

   Plasmid (PSB1C3-RFP) ----------------- 2 μL
   TorCAD (BBa_K1086000) ---------------- 2.5 μL
   Buffer 10X --------------------------- 1 μL
   T4 DNA Ligase ------------------------ 1 μL
   ddH2O (di-deionized) ----------------- 3.5 μL

• Incubate it overnight at 4ºC.

Transformation Protocol (as suggested by iGEM-registry)

• Start thawing the chemically competent cells on ice.
• Add 1 - 2 μL of DNA to the 2 mL tube. Pipet up and down a few time, gently. Make sure to keep the competent cells on ice.
• Close the tubes and incubate the cells on ice for 30 minutes.
• Add cells tubes by immersion in a preheated water bath at 42 °C for 60 seconds.
• Incubate the cells on ice for 5 minutes.
• Add 400 μL of 2xYT media (make sure that the broth does not contains antibiotics and is not contaminated) to each transformation.
• 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.

• Label two petri dishes with 2xYT agar (AMP or CHL). Plate 20 μL and 200 μL of the transformation onto the dishes, and spread. This helps you ensure that you will be able pick out a single colony.
• 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.
• Pick a single colony, make a glycerol stock, grow up a cell culture and miniprep.

Miniprep

- We used Promega (Wizard® Plus SV Minipreps DNA Purification Systems) and Invitrogen (PureLink™ Quick Plasmid Miniprep Kit) kits. We followed manufacturer’s indications.

PCR Protocol

   Compound ------------------------ Volume
   ddH2O --------------------------- 8.0 μL
   Buffer IB 10x ------------------- 1.5 μL
   dNTP’s 2.5 mM ------------------- 1.5 μL
   Primer VF2 10 uM ---------------- 0.4 μL
   Primer VR 10 uM ----------------- 0.4 μL
   Taq 5u/ μL ---------------------- 0.2 μL
   DNA ----------------------------- 3.0 μL
   Final volume -------------------- 15 μL

Amplifications were performed in M.J. Research PTC-100 (GMI Inc.) thermocyclers. The amplification program was as follows:

• 10 minutes of initial denaturation at 94ºC.
• 30 cycles of denaturation (94ºC for 1 minute), annealing (55ºC for 1 minute) and extension (72ºC for 1 minute).
• 10 minutes of final extension at 72ºC.

- PCR products were analysed in 1% agarose gels, using 1Kb DNA Ladder (Invitrogen) as molecular weight ladder. Gels were stained with Sybr Safe (Life Technologies).

Fluorometric assay for RCNA-YFP activation (Protocol # 1)

• Measure OD600 of cultures to be assessed.
• Dilute cultures to OD600 = 0.02, in a volume of 3 mL.
• Prepare a solution of CoCl2 1 mM, using 6 μL of 250 mM solution (see protocol 3) and 1494 μL of 2xYT media.
• Make other 6 cobalt solutions using the mixture on step (3).

50 uM -> 50 μL of 1 mM solution + 950 μL of media 2xYT. 100 uM -> 100 μL of 1 mM solution + 900 μL of 2xYT media. 150 uM -> 150 μL of 1 mM solution + 850 μL of 2xYT media. 200 uM -> 200 μL of 1 mM solution + 800 μL of 2xYT media. 250 uM -> 250 μL of 1 mM solution + 750 μL of 2xYT media. 300 uM -> 300 μL of 1 mM solution + 700 μL of 2xYT media.

• In a 96 well plate, add 100 μL of the cultures from step (2) in each well, in triplicate.
• Make the blank: add 100 μL of media 2xYT in another wells, to discard the noise from media and cobalt.
• Add 100 μL of cobalt solutions from step (4) to cultures and to the blank:

- To a final concentration of 25 μM, use 100 μL of the 50 μM cobalt solution;

- To a final concentration of 50 μM, use 100 μL of the 100 μM cobalt solution;

- To a final concentration of 75 μM, use 100 μL of the 150 μM cobalt solution;

- And so on….

• Seal the plate.

• Read fluorescence: 514 nm (excitation) and 527 nm (emission), every 15 minutes, for 16 hours.

Note: It is not recommended to read absorbance using black plate, as it causes interference on reading, despite it being usually utlized for fluorimetric assay.

Fluorometric assay for RCNA-YFP activation (Protocol # 2)

• Measure OD600 of bacterial cultures.
• Dilute cultures to OD600 = 0.1.
• Let bacteria grow until OD600 = 0.5.
• Proceed as protocol #9, from steps 3 to 8.
• Read absorbance at 600 nm and fluorescence at 514 nm (excitation) and 527 nm (emission), every hour, until 4 hours after the first read. Let bacteria grow for more 4 hours and then read absorbance and fluorescence every 2 hours, for 16 hours.

Hindlimb ischemia model

Figure 1. Mice were subjected to unilateral permanent left femoral artery occlusion (FAO), as described by Madedu and colleagues (2006, 2008), under anesthesia with xylazine (10 mg/kg) and ketamine (100 mg/kg) i.p.. (Hindlimb ischemia)

Figure 2. Using a stereoscopic microscope, the set artery-vein-femoral nerve was exposed and the femoral artery dissected, occluded with two nodes (Hindlimb ischemia)

Figure 3. And then the region between nodes were electrically coagulated. Then the animals had the incisions sutured and were kept under artificial heating until complete recovery by three days when occur the peak of inflammation and the mices can be bleed.(Hindlimb ischemia)

(Hindlimb ischemia)

(Hindlimb ischemia)

• The blood was donated by Angiogenesis Laboratory from Biological Science Institute in UFMG where it was collected from brachial plexus of the mice according to ethic committee approval from CETEA-UFMG, licence 253/08.
• The Serum obtained was centrifuged by 10 minutes 4000 rpm in room temperature.

References

• Madeddu P; Emanueli C; Spillmann F; Meloni M; Bouby N; Richer C; Alhenc-Gelas F; Van Weel V; Eefting D; Quax PH; Hu Y; Xu Q; Hemdahl AL; van Golde J; Huijberts M; de Lussanet Q; Struijker Boudier H; Couffinhal T; Duplaa C; Chimenti S; Staszewsky L; Latini R; Baumans V; Levy BI. Murine models of myocardial and limb ischemia: diagnostic end-points and relevance to clinical problems.Vascul Pharmacol. 2006;45:281-301.

• Madeddu P; Kraenkel N; Barcelos LS; Siragusa M; Campagnolo P; Oikawa A; Caporali A; Herman A; Azzolino O; Barberis L; Perino A; Damilano F; Emanueli C; Hirsch E. Phosphoinositide 3-kinase gamma gene knockout impairs postischemic neovascularization and endothelial progenitor cell functions. Arterioscler Thromb Vasc Biol. 2008 (1):68-76.

Image References

• Niiyama H, Huang NF, Rollins MD, Cooke JP. Murine model of hindlimb ischemia. J Vis Exp. (JoVE) 2009 Jan 21;(23). doi:pii: 1035. 10.3791/1035.

• Limbourg A, Korff T, Napp LC, Schaper W, Drexler H, Limbourg FP. Evaluation of postnatal arteriogenesis and angiogenesis in a mouse model of hindlimb ischemia. Nat Protoc. 2009;4(12):1737-46. doi: 10.1038/nprot.2009.185.

IMA binding assay: test of bacteria with RCNA-YFP using mice serum

• Follow the steps 1 to 3 from Protocol #10
• Prepare a cobalt (CoCl2) solution in culture media at 100 mM. This is the blank solution. Distribute it in 7 wells.
• Distribute 100 μL of media with bacteria in 12 wells. Add 100 μL of mice sera (3 serum for control and 3 serum with ischemia), in duplicates.
• For the positive control, add media with cobalt instead of the serum.
• Do the same as step #5 but adding the ischemic mice serum.
• Read fluorescence at 514 nm (excitation) and 527 nm (emission) every hour during 16 hours.

BSA-Cobalt binding assay

• Follow the steps 1 to 3 from Protocol #10
• Prepare a solution of cobalt (CoCl2) in culture media at 100 mM. Use this is as blank solution
• Prepare the solution of BSA at 66 mg/ml in media with cobalt. Make a serial dilution to obtain different BSA concentrations: 66 mg/ml, 33 mg/ml, 16.5 mg/ml, 8.25 mg/ml, 4.125 mg/ml, 2.063, mg/ml 1.032 mg/ml, 0.516 mg/ml, 0.258 mg/ml, 0.129 mg/ml, 0.065 mg/ml, 0.033 mg/ml, 0.017 mg/ml, 0.009 mg/ml, 0.005 mg/ml, 0.003 mg/ml
• Make triplicates for each BSA solution earlier made in step 3.
• In each well, add 100 uL of bacteria culture obtained in step 1.
• Seal the plate.
• Read fluorescence at 514 nm (excitation) and 527 nm (emission) every hour during 16 hours.

Fluorometric assay for TorR+RFP activation

• Proceed as steps 1 to 3 from protocol #10.
• Prepare a solution of TMAO in media with a concentration of 600 mM. Make a serial dilution, to obtain different TMAO concentrations: 300 mM, 200 mM, 100 mM, 10 mM, 1 mM, 100 μM, 10 μM, 1 μM
• Prepare the blank solution with culture media and TMAO. And pipette it on 7 wells.
• Pipette the TMAO solutions with different concentrations, making triplicates for each one.
• Add 100 μL of bacteria culture obtained in step 1 and add it in each well from step 4.
• Seal the plate. Read fluorescence at 587 nm (excitation) 610 nm (emission).

Fluorometric Data Analysis

• To leading with an anisotropyc effect observed with all of our experiments using YFP we treated the data as follow:

• For the results using red fluorescent protein we used :

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