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From 2011.igem.org

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Protocols

This page lists all the protocols used in our project. We have classified them into five main categories as follow.

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Gene Guard

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Genome integration

- Grow a culture of CRIM helper plasmid containing cells and make them competent.

- Transform competent cells with CRIM plasmid.

- After transformation, suspend cells in SOC without ampicillin.

- Incubate at 37°C for one hour.

- Incubate at 42°C for 30 minutes.

- Plate cells on agar plates containing kanamycin.

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Soil survivability

- Grow cultures of E. coli expressing your favourite reporter gene.

- Autoclave soil in a 100 ml Schott bottle to make it sterile.

- Collect non-sterile soil from the same source in another bottle

- Fill 1.5 ml Eppendorf tubes with about 1 ml of sterile or non-sterile soil.

- Hole-punch filter paper to create small discs that you subsequently autoclave.

- Dip the filter papers into your bacterial media and put one filter paper in each Eppendorf.

- For the negative control, put non-inoculated filter discs into Eppendorfs containing non-sterile soil.

- Keep the Eppendorfs at room temperature.

- Each week, grow up a sample each from the sterile soil, non-sterile soil and control Eppendorfs. For this, remove the filter disc and put it in 1 ml of LB containing the selective antibiotic. Grow for three or four hours into exponential phase, then plate about 50 μl of each culture onto an LB plate containing the selective antibiotic. Grow overnight and check for fluorescence the next day using a blue light box or UV imager (or whatever else is appropriate for the reporter you are using).

To test for presence of the insert:
- Pick a colony from each plate (or several if you can see different colony morphologies on one plate) and grow them up in LB containing selective antibiotic

- Perform a miniprep on each of the cultures (see our cloning protocols section)

- Digest with EcoRI and PstI (or whatever other restriction enzymes you can use to remove your reporter gene from its vector) for 90 minutes according to the manufacturer's instructions, then either heat inactivate or immediately run on a 1% agarose gel

To determine bacterial species
- Set up a colony PCR (see cloning protocols) with the following primers:

515F (GTGCCAGCMGCCGCGGTAA, M=A or C)

1492R (GGYTACCTTGTTACGACTT, Y=C or T)

27F (AGAGTTTGATCMTGGCTCAG)

- These are degenerate primers that you can order separately and combine before setting up the PCR reaction (primer sequences from Tanner M et al. (2000) Molecular phylogenetic evidence for noninvasive zoonotic transmission of Staphylococcus intermedius from a canine pet to a human. Journal of Clinical Microbiology 38(4): 1628-1631.)

- Either dip the colonies into sterile water for ten minutes or dip them directly into the PCR mix and add an initial boiling step at 95°C for ten minutes on the PCR machine

- Run 35 repeats of the reaction for 20 seconds at 95°C, 20 seconds at 50°C and 90 seconds at 72°C, followed by a final extension step of five minutes at 72°C.

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Plasmid retainment

- Pick a colony of E. coli expressing a reporter gene. (Our experiment was performed using sfGFP (BBa_K515105). Inoculate the cells into 5 ml LB with no antibiotic.

- Next day take 50 μl of culture grown overnight and reinoculate into fresh LB with no antibiotic and grow overnight.

- From the overnight culture also take 50 μl and spread it on an agar plate containing antibiotic corresponding to the resistance present on the plasmid with the reporter gene. Grow the cells and image them next day. Imaging was done using Fujifilm LAS - 3000 Imager.

- Repeat the reinoculation, growth on plate with antibiotic and data collection for a number of days to observe the plasmid retainment over time.