From 2007.igem.org

Filter Conjugation Protocol

We are using conjugation as a means of transferring a plasmid from transformed E.coli into S.oneidensis. The plasmid contains kanamycin resistance, and the S.oneidensis have gentamicin resistance. Thus, a double selection is used to select for S.oneidensis with plasmid (resistant to both kanamycin and gentamicin). During this double selection, the gentamicin will kill any E.coli and the kanamycin will kill S.oneidensis that lacks plasmid.

Add 0.75mL of magnesium sulfate (MgSO4) to a centrifuge tube filter.

Add 9.3 microliters of the donor cells (E.coli BW20767) and 9.3 microliters of the acceptor cells (S.oneidensis AS-92) to the centrifuge tube filter.

Centrifuge until all of the MgSO4 elutes from the column through the filter.

The filter is now covered with the condensed pellet of cells (thus encouraging conjugation)

Remove the filter from the centrifuge tube filter with sterile tweezers.

Incubate the filter (cell-side up) on an LB agar plate for at least 5 hours at 30 degrees C.

Add 1 mL of LB broth to a tube.

Add the filter (now with grown out cell colonies) into the LB broth.

Vortex the tube to release the cell colonies from the filter surface.

Plate the liquid cell stock onto a double selection(kanamycin and gentamicin) plate.

(DS)

Calcium Chloride/Heat Shock Plasmid Transformations Protocol

Reagents to be Supplied by the User

LB plates with appropriate antibiotic concentration (for pET -25b(+), amipicillin at 50-100 microgram/mL) SOC media

1) Prepare one LB-Amp plate for each transformation, plus one plate for a negative (no plasmid) control. After storage at 2-8 degrees C, equilibrate to room temperature.

2) Centrifuge the tubes containing plasmid DNA to collect contents at the bottom of the tube. Add 1 microliter DNA to a sterile 1.5 mL tube on ice. Have one tube on ice with no DNA.

3) Add 50 microliters of competent cells (either freshly prepared, or frozen and thawed on ice). Avoid excessive pipetting, as cells are very fragile.

4) Gently flick the tubes to mix, and place on ice for 20 minutes.

5) Heat shock the cells for 45 seconds to 2 minutes in a water bath at exactly 42 degrees C. Do not shake.

6) Immediately return the tubes to ice for 2-10 minutes.

7) Add 950 microliters of room temperature SOC media to the tubes.

8) Incubate 1-1.5 hours at 37 degrees C with shaking (~150 rpm).

9) Plate 100 microliters of each transformation culture onto antibiotic plates.

10) Incubate the plates overnight (16-24 hours) at 37 degrees C.

See Making Heat Shock Competent Cells for more information. "

Negative Control: No plasmid

Trial 1:

We followed this protocol from step 3 in order to practice our transformation and conjugation techniques. The competent cells given to us by Joshua were E.coli sm10; the GFP plasmids (pMS291/lacI) were supplied by Ilaria. But we performed the protocol with four samples of plasmid DNA+E.coli. Each sample contained 1 microliter of 63.5 ng/microliter GFP plasmid DNA; 50 microliters of the competent E.coli cells; and 950 microliters of SOC media. 100 microliters of each sample tube were plated onto one kanamycin plate and one blank (non-antibiotic), control plate.

Trial 1 results:

A lawn of bacteria on each plate. No GFP fluorescence under UV illumination.

Trial 2:

Used E.coli sm10 cells, GFP plasmid pMS291 with lacZ promoter (stimulated by IPTG).

Sample 1 contained 50 microliter sm10 + 1 microliter mPS291.

Sample 2 contained 50 microliter sm10 + 1 microliter water.

Sample 3 contained 50 microliter E.coli Dh5-alpha + 1 microliter water.

Samples 2 and 3 served as negative controls (should show no colonies in the presence of kanamycin).

5 microliters of each of these samples were plated onto [LB]+[20microliters of 50X kanamycin]+[20microliters of 100micromolar IPTG].

50 microliters of each of these samples was also plated onto [LB]+[20microliters of 50X kanamycin]+[20microliters of 100micromolar IPTG].

Trial 2 results:

Colony growth on all plates. So the kanamycin is not killing any of the cells. Perhaps the kan plates were made incorrectly.

Trial 3:

Repeated Trial 2 but with double volume of kanamycin and double volume of IPTG (to ensure stimulation of lacZ promoter an thus GFP expression).

Trial 3 results:

5 microliters sm10 + water in IPTG and kan: lawn of colonies

50 microliters sm10 + water in IPTG and kan: less than 10 colonies

5 microliters sm10 + pMS291 in IPTG and kan: less than 10 colonies, fluorescent under UV illumination !

50 microliters sm10 + pMS291 in IPTG and kan: less than 10 colonies, no fluorescence

5 microliters Dh5-alpha + water in IPTG and kan: more than 100 colonies

5 microliters sm10 + water in kan: lawn of colonies

5 microliters sm10 + pMS291 in kan: lawn of colonies

5 microliters Dh5-alpha + water in kan: more than 100 isolated colonies

E.coli successfully transformed with pMS291 (showed fluorescence). These colonies will be grown out in SOC media for further examination. But kan still seems to be innefective.

[update] The succesfully transformed E.coli that fluoresced were grown out in SOC media and then centrifuged to obtain a pellet of cells. This pellet definitely fluoresced under UV confirming that the transformation was succesful.

Trial 4:

Two stocks were made: one with 20 microliters E.coli Dh5-alpha + pMS291 (lacZ), and the second with 20 microliters E.coli Dh5-alpha + water. Each of these two stocks were plated onto 4 different plates.

Plate 1 contained LB

Plate 2 contained LB + kanamycin

Plate 3 contained LB + kanamycin + IPTG

Plate 4 contained LB + kanamycin which was dripped onto the LB

Trial 4 Results:

20 microliters E.coli Dh5-alpha + pMS291 (lacZ)

Plate 1 containing LB: lawn of bacteria

Plate 2 containing LB + kanamycin: more than 100 colonies

Plate 3 containing LB + kanamycin + IPTG: more than 100 colonies and fluorescent!

Plate 4 containing LB + kanamycin which was dripped onto the LB: more than 100 colonies

20 microliters E.coli Dh5-alpha + water

Plate 1 contained LB: no colonies

Plate 2 contained LB + kanamycin: no colonies

Plate 3 contained LB + kanamycin + IPTG: no colonies

Plate 4 contained LB + kanamycin which was dripped onto the LB: no colonies

Therefore, the kanamycin worked correctly. E.coli without pMS291 (without kanamycin resistance) all died. Transformation was also successful

(DS)

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