Luria Bertani

E.coli K12 TGI were grown to log phase (up to OD6oo=0.20-0.30) in Luria-Bertani (LB) broth, washed and ultimately concentrated 25 times in ice-cold 100 mM of CaCb. DNA was extracted from agarose gel after electrophoresis, added to 200 ml of competent cell and incubated at 0°C for 15 min. The cell-DNA complex was transferred to 42°C for exactly 90 s and was rapidly chilled in ice. Then 1000 ml LB-broth was added and the cells were incubated at 37°C for 60 min. 100 ml cells was spread on LB-agar with and without selective marker ampicillin (50 mg/ml), to obtain the number of transformants and viable cells respectively. Plates were incubated at 37°C for 18-24 h. 

Luria-Bertani (LB) liquid medium and LB agar plates containing ampicillin... 

Luria-Bertani (LB) medium tryptone (25 g), yeast extract (13 g), NaCl (25 g)... 

E. coli BL21(DE3) harboring plasmid pPV2.85 (frozen glycerol stocks) Luria-Bertani (LB) broth powder (20 g L )... 

Tryptone (5 g), yeast extract (2.5 g) and NaCl (5 g) were dissolved in distilled water, the volume was adjusted to 500 mL and then autoclaved (20 min, 120 °C). A small portion of this Luria-Bertani (LB) medium (10 mL) was placed into a sterile 100 mL shake flask and ampicillin and chloramphenicol solutions were added (LBamp+cm) to final concentrations of 100 p,g mL and 20 p.g mL respectively. The solution was inoculated with E. coli JM109 pGro7 pJOE4072.6 and shaken overnight at 37 °C and 200 rpm. This overnight culture (2 mL) was used to inoculate 200 mL LBamp+cm in a... 

Luria-Bertani (LB) medium (tryptone peptone 10 g yeast extract, 5 g L , NaCl 5 g... 

Luria-Bertani (LB, 1.0% polypeptone, 0.5% yeast extract, 1.0% NaCl, pH 7.0) medium containing 100 pg/mL ampicillin (LB-amp) and LB-amp agar medium (LB containing... 

LB apiarplate. Luria-Bertani (LB) medium (Difco, NJ, USA) and Bacterial agar (Difco). 

Luria-Bertani (LB) medium. Each liter contains 10 g Bacto-tryptone, 5 g Bactoyeast extract, and 10 g NaCl. Adjust to pH 7.5 with NaOH. Ampicillin. A solution of the sodium salt is prepared in water (25mg/mL) and sterilized by passage through a microfilter (e.g., a 0.22 H Millipore filter). Store the solution in a freezer. 

Luria-Bertani (LB) solid or liquid medium and resuspend the pellet in 576 fih Milli-Q water. Incubate the samples for lh at 37°C with proteinase K (20 gg/gL) and SDS 10% (p/v) (pH 7.2). Extract twice with phenol-chloroform-isoamyl alcohol (25 24 1) and collect DNA by precipitation with isopropanol. Resuspend it in 100 gL Milli-Q water. 

Select single colonies and grow overnight at 250 rpm, 37°C in 5 mL Luria-Bertani medium supplemented with ampicillin at 100 pg/mL (LBamp). 

E. coli XLl-Blue was grown at 37°C in Luria-Bertani (LB) medium (containing 10 g/L of tryptone, 5 g/L of yeast extract, and 5 g/L of NaCl). Recombinant E. coli strains for the PHA production were cultivated at 30°C for 72 h in LB medium containing two different carbon sources (1) 2 g/L of sodium decanoate (Sigma, St. Louis, MO) and (2) 10 g/L of sodium gluconate (Junsei, Tokyo, Japan) plus 2 g/L of sodium decanoate. All the flask cultures were carried out in triplicate in a rotary shaker at 250 rpm. For the cultivation of recombinant E. coli strains, ampicillin (50 mg/L) was added to the medium. 

Luria-Bertani (LB) agar, Mueller-Hinton (MH) agar or M9 agar (all from Difco Laboratories). The choice of the medium depends on the peptide used. If the peptide s activity is salt-sensitive, use of a low-salt minimal medium such as M9 is more appropriate. 

Kinase insert domain-containing receptor KDR is the hnman homolog of the monse FLK-1 receptor the KDR and FLK-1 receptors are also known asVEGFR2 seeVEGFR Mast/stem cell growth factor receptor, CD 117 Retroviral oncogene derived from HZ4 feline sarcoma Kruppel-like factor 5, a transcription factor Lymphokine-activated killer cells Linear-after-the-exponential-PCR Luria-Bertani... 

A 10 mmol L stock coelenterazine solution was prepared by dissolving coelenterazine (Nanolight Technology, Prolume Ltd. Pinetop, AZ, USA) in methanol for use at a final concentration of 10 /tmol L". All coelenterazine solutions were stored at -20 °C and working solutions were kept on ice in the dark during preparation. Diluent buffers comprised distilled water (dH20), Phosphate buffered saline (PBS), Buffer A (10 mmol L Tris [pH 7.8], 1 mmol L EDTA, 0.6 mol L NaCl), 7H9 medium supplemented with Tween-80 with or without 10% OADC (oleic acid, albumin, dextrose, catalase), Luria-Bertani (LB) broth with or... 

Bacterial strains and growth conditions. Escherichia coli DH5a, E. coli JM109 were purchased from TaKaRa company and used in this study, E.coli W3110 was kindly presented by professor Zhou of Academy of Military Medical Sciences of China. Strains were routinely grown in Luria-Bertani media at 37 °C for 24 h. 

Cloning and sequencing of amplicons. The amplified products were cloned into the pGEM-T vector (Promega) and used to transform competent E. coli DH5a. Transformed E. coli DH5 was then plated onto Luria-Bertani agar with 50 pg/mL... 

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