"

From 2012.igem.org

Identification of BADmeat promoters by transcriptome analysis

Introduction to the MicroArray technique

This technique encompasses always a comparison between two conditions. Can be a knock-out strain and WT, but also the same strain on a different “diet”. In our case, Bacillus was subjected to volatiles of fresh vs. bad meat at a certain OD, in our case: OD 0.8-1.0, late exponential phase because this should yield much RNA. One isolates all mRNA (the transcriptome) and with reverse transcriptase, the RNA is copied into cDNA. The cDNA is tagged with a fluorescent dye, one condition red, the other green. Both conditions are then brought onto a MicroArray slide, where DNA of each gene of the organism of interest (in our case, Bacillus) is attached in separate wells (these are called “probes”). When the cDNA is brought onto the slide, it will hybridize with the probes. After fluorescence measurement, one will see a black dot when the gene is not transcribed in both conditions, an orange/yellow one when the gene is transcribed in both, and a green or red one when just one condition causes the transcription of the gene. The ratio of red/green will be a measure for differential expression.

Sensing volatiles with Bacillus subtilis

How to measure the transcriptome of Bacillus subtilis subjected to meat volatiles? Bacillus should be able to grow without many restrictions, as much volatiles as possible should get into the meat. --> tried a few setups (also fermentors). In the end: a simple closed system, with a pot with rotten meat (rotten in fridge for 14 days), from which the air (filtered) goes trough Bacillus in LB medium, stirred with a magnetic stirrer. The air is pumped through the system with a simple peristaltic pump.

Growth conditions Bacillus: volatiles/oxygen depletion

Apart from the micro-array, we’ll do this experiment again measuring the OD to see if there is a difference in growth rate for Bacillus.

Identification of rotten meat volatiles

The gas chromatography should identify the different volatiles present in the rotten meat. If it is possible, it also should give quantitative data, however, this is difficult due to the probably large diversity of volatiles present.