Talk:IGEM:MIT/2006/Notebook/2006-6-14

Negre-ArchBiochemBiophys-2002
Some random notes measuring products...

Materials and methods
"Product verification was performed by growing BL21 (DE3) cells expressing SAMT and those containing pET-28a vector (controls) in the presence (5 &mu;g/ml) and absence of salicylic acid and benzoic acid under the conditions described above. After the cells were harvested by centrifugation, the cultured medium (25 ml) was extracted with 5 mL of hexane, and the hexane phase was concentrated to 200 μl and analyzed by gas chromatography–mass spectrometry [ Dudareva-PlantCell-2000 ]." Negre-ArchBiochemBiophys-2002

Results and discussion
"Moreover, the culture medium of the E. coli cells expressing SAMT contained methyl salicylate (2.1 &mu;g/ml) (Fig. 2B) when the growing medium was supplemented with 5 &mu;g/ml salicylic acid, and methyl benzoate (0.86 &mu;g/ml) when the growing medium was supplemented with 5 &mu;g/ml benzoic acid (Fig. 2C). E. coli cells that contained a pET-28a plasmid without the SAMT coding region did not have any detectable enzyme activity and did not produce methyl salicylate or methyl benzoate (Fig. 2D)." Negre-ArchBiochemBiophys-2002

Figure 2 caption
"A) Gas chromatography–mass spectrometry analysis of methyl salicylate and methyl benzoate standards. Electron impact mass spectrum of methyl benzoate is shown in the upper left-hand corner and of methyl salicylate in the upper right-hand corner. Numbered peaks represent mass-to-charge ratios of molecular ions and fragment ions of methyl benzoate and methyl salicylate. (B) Analysis of the medium of E. coli cells expressing snapdragon SAMT after induction with isopropyl &beta;-Image-thiogalactopyranoside when the growing medium was supplemented with 5 &mu;g/ml salicylic acid. The mass spectrum is that of the peak eluted at the same retention time as the authentic methyl salicylate standard (A). (C) Analysis of the medium of E. coli cells expressing snapdragon SAMT after induction with isopropyl &beta;-Image-thiogalactopyranoside when the growing medium was supplemented with 5 &mu;g/ml benzoic acid. The mass spectrum is that of the peak eluted at the same retention time as the authentic methyl benzoate standard (A). (D) Analysis of the medium of E. coli cells expressing pET-28a vector with no insert after induction with isopropyl &beta;-Image-thiogalactopyranoside. Indole is produced by all E. coli cells." Negre-ArchBiochemBiophys-2002

Materials and methods
Note that this refers to compounds collected directly from plants.

"Trapped floral scent compounds were analyzed by gas chromatography–mass spectrometry (GC-MS) with a FinniganMAT GCQ instrument (Thermoquest, San Jose, CA) (injector temperature 230&deg;C, injector volume 1 &mu;L, and split ratio 50:1) using a DB-1 nonpolar capillary column (30 m x 0.25 mm [i.d.]; film thickness 0.25 &mu;m). Ionization energy was set at 70 eV. Column temperature was held at 50&deg;C for 1 min and then heated to 240&deg;C at 10&deg;C min-1. The mass spectrometer was scanned from 41 to 400 amu. Ambient volatiles collected at the same times as the samples were used as controls. Components were first identified from a computer database containing several thousand mass spectra and were confirmed by comparing retention times and mass spectra with those of authentic standards." Dudareva-PlantCell-2000

On extraction of the scent compounds from the medium ...

"Extraction of Methyl Benzoate from the Medium of E. coli Cells and GC-MS Analysis

BL21 (DE3) cells expressing BAMT and those containing pET-T7 (11a) vector (controls) were grown in the presence (5 &mu;g mL-1) or absence of benzoic acid under the conditions described above. After the cells were harvested by centrifugation, the culture medium (25 mL) was extracted with 5 mL of hexane, the hexane phase was concentrated to 200 &mu;L and analyzed by GC-MS ( Dudareva-PlantJ-1998 ). " Dudareva-PlantCell-2000

Results
"Expression of BEAT cDNA in E. coli

To further verify that the cDNA we isolated encodes BEAT, we cloned it into the pET-T7(11a) expression vector, transformed E. coli cells with the recombinant plasmid, and induced the expression of this foreign gene. E. coli expressing C. breweri BEAT contained large amounts of a protein with apparent molecular mass on SDS–PAGE of 58 kDa, the same as the plant-purified BEAT, both in insoluble inclusion bodies ( Fig. 1) and in soluble form (data not shown). This protein had a very similar enzymatic activity to that of plant BEAT ( Fig. 2c and Table 1). E. coli cells harboring a pET-T7(11a) plasmid without the BEAT coding region did not have any BEAT activity, nor did bacteria lacking this plasmid entirely (data not shown). Furthermore, the spent medium in which E. coli cells expressing BEAT grew contained significant amounts of benzylacetate (2 &mu;g ml-1) ( Fig. 2d), whereas bacterial cells not containing the recombinant plasmid did not ( Fig. 2e). Both types of cultures produced copious amounts of indole. It should be noted that the cells were grown in regular LB medium (containing the antibiotic ampicillin) without the addition of any benzylalcohol." Dudareva-PlantJ-1998

Experimental procedures
"Expression of BEAT in E. coli

The coding region of BEAT was amplified with the forward 25-mer oligonucleotide 5'-CCATATGAATGTTACGATGCACTCC-3', and the backward 34-mer oligonucleotide 5'-TGGATCCTTAGGAAACGTATGAAAGCAGTTGGTG-3'. The forward oligonucleotide introduced an NdeI site at the initiating methionine ATG codon. The backward nucleotide introduced a BamHI site downstream of the stop codon, and also eliminated the single original NdeI site in BEAT cDNA which occurs 6–11 nucleotides upstream of the stop codon. The elimination of this NdeI site was accomplished by changing the original T at the third position of the codon for tyrosine 431 to C, thus maintaining a tyrosine codon. The PCR-amplified 1.3 kb fragment was cloned into the NdeI–BamHI site of the expression vector pET-T7(11a), the resulting plasmid was transferred into E. coli BL21 cells, and the expression of BEAT cDNA was induced by the addition of 0.4 m m IPTG at A600 of 0.5 with 3 h incubation at 37°C ( Wang et al. 1997). Cell cultures (25 ml) were harvested by centrifugation, resuspended in 100 m m NaCl, 50 m m Tris–HCl pH 8.0, 1 m m EDTA and 1 m m PMSF, and sonicated on ice with a microtip probe for four time intervals of 30 sec each. After spinning this lysate at 12 000 g for 5 min, soluble and insoluble fractions were assayed for enzyme activity and analyzed by SDS–PAGE. The E. coli-expressed BEAT protein was further purified by DE53 anion exchange and Mono-Q chromatography.

Extraction of benzylacetate from E. coli cells and GC–MS analysis

BL21 cells expressing BEAT and those without the pET-T7(11a)–BEAT plasmid were grown under conditions described above. After harvesting the cells for protein purification, the spent medium (25 ml) was extracted with 5 ml of hexane, and the hexane phase removed, placed in a glass tube, and reduced to 0.2 ml by passing N2 at the opening of the tube. The samples (3 &mu;l of the hexane concentrate) were analyzed by GC–MS." Dudareva-PlantJ-1998

Things to get

 * Access to GC-MS
 * Methyl salicylate (standard) - Brian Cook has some we could use.
 * Methyl benzoate (standard)
 * Indole (control)
 * Hexane? - TK found some Heptane. Should work.