Transferring pBE110 to S17-1

May 29, 2014

Struck out fresh E. coli S17-1 onto LB only agar. Incubated at 37C overnight.

Brothed isolated colony of pBE110 (in T1R E. coli) into 10 mL of LBTet15. Incubated at 37C, shaking at 225 rpm.

May 30, 2014

Performed plasmid isolation of pBE110 using Zyppy kit. Purified plasmid DNA using Zyppy DNA clean + concentrator kit.

Made electrocompetent  S17-1 E. coli cells from the plate made yesterday. Performed procedure on May 21, 2014 to make electrocompetent cells.

Electroporate transformed pBE110 into the electrocompetent S17-1 cells. Used pUC19 as control. Immediately after electroporating, added 1 mL of warmed SOC media and incubated at 37C for 1 hour. Plated pBE110 onto LBTet15Tm200 and pUC19 onto LBAmp100. Incubated at 37C overnight. Will check if have any colonies tomorrow.


//EWW

RNA Extraction

May 29, 2014

DeeptiT. of the T.Bergholz lab checked the purity of the extracted RNA from the two S. maltophilia isolates. The results below are of the extracted nucleic acids without DNAse being added yet.

Isolate	260	280	260/280	260/230	ng/ml
B24	58.0	29	2.00	1.97	2320.7
B84	47	23.5	2.00	1.94	1824.0

Purity and concentration look excellent. However, this is before DNAse was added, so we could still be detecting DNA in the sample. DNAse will be added and samples will be read again in the near future.

Transferring pBE110 to S17-1

Confirmed that S17-1 is indeed resistant to trimethoprim antibiotic at 200 ug/mL concentration. Previously brothed isolated colony of S17-1 in LBTm200 broth. Incubated at 37C 225 rpm overnight and had growth the next morning. DH5a control checked out as well.

Last attempt to transfer pBE110 to S17-1 was unsuccessful (May 23, 2014). However, the pUC19 control was successful and colonies were found on that plate. Meaning that there maybe is an issue with the pBE110 DNA that I am using. It is likely not pure or at a high enough concentration. I will purify my sample and attempt transformation again in the future.

RNA Extraction

Shadowed an RNA Extraction method (procedure not shown) with DeeptiT. of the T.Bergholz lab. Attempted to extract RNA from two S. maltophilia strains (B2423 and B84). This method doesn't make use of a kit or kit reagents, so if successful this method could possibly be included in a future methods paper for S. maltophilia.

Bacterial strains were grown to stationary phase in 5 mL of LB broth at 37C 225 rpm before beginning the procedure.

Nucleotide samples were stored at -80C overnight before DNAse treatment tomorrow.

Transferring pBE110 to S17-1

Repeated procedure on May 20, 2014. Made fresh LB agar plates with Tet15 and Tm200.

Brothed Stenotrophomonas maltophilia B24 and B84 into 5 and 10 mL of LB broth and incubated at 37C 225 rpm. Will attempt to extract RNA from steno species tomorrow. Also, will implant inoculated catheter into dubia roach tomorrow.

//EWW

Transferring pBE110 to S17-1

May 20, 2014
Created electrocompetent E. coli S17-1 cells using cold treatment. The goal is to transfer the pBE110 to this E. coli strain because it can more readily conjugated into Stenotrophomonas and other bacterial strains we are interested in studying. Right now, we only have the pBE110 in T1R E. coli cells (the chemically competent ones).


Making Electrocompetent Cells:

Note: Put sterile nuclease free water on ice to chill.

1. Streak culture onto appropriate media and incubate overnight2. Re-suspend one loop full of bacteria in 500 uL of ice-cold ddH₂O in 1.5 m centrifuge tubes (2-3 day old plates work best) 3. Spin to pellet (24,000 x g for 1 minute 4° C)4. Discard supernatant5. Re-suspend pellet in 500 uL of ice-cold ddH₂O6. Repeat steps 3 – 57. Repeat steps 3 - 48. Re-suspend pellet in 50 uL of ice-cold ddH₂O

Electroporation

Note: Put SOC media into 37C to warm it up before use in recovery after electroporation. Put cuvettes on ice to chill them. Ideally, pipets should be put in fridge to chill them as well.

      Controls : Added 1 uL of pUC19 DNA to 49 uL of competent S17-1 and put in a chilled 1 mm disposable electroproation cuvettes (Link). Added only 50 uL competent S17-1 cells to chilled cuvette.

     Experiment: Added 5 uL of pBE110 DNA to 45 uL of competent S17-1 cells. Transferred to chilled electroporation cuvette. Repeated this treatment group in triplicate.

1. Placed cuvettes into Bio-Rad MicroPulser Electroporator (Link) and ran per the manufacturer's instructions using the "Ec1" program at 1.8 kV for one pulse.

2. Immediately added 1 mL of warmed SOC media to each cuvette after electroporation

3. Incubated cuvettes at 37C static for 1 hour
       Note: put selective abx media in 37C to warm up media

4. Spread plated 200 uL of cuvette broth for each treatment onto appropriate antibiotic media. pUC19 will be plated onto LB Amp100. pBE110 experimental treatment will be plated onto LB Tet15 Tm200 (Tm = trimethoprim)
      Note: used 200 uL instead of the traditional 100 uL because I didn't have very high hopes of getting colony growth

5.  Incubated at 37C overnight.

May 21, 2014

Results:

Only had growth on the pUC19 growth plate (4780 CFU/mL). There was no colony growth on any of the pBE110 experiment treatment. The agar plates that were used for the experimental treatment were a month old, and there was some question about what antibiotics were actually on the agar. The markings on the plate contradicted the label on the bag.

Future:

Will make new agar for experimental treatment so I know that the antibiotics are correct. Then will repeat this experiment.

Creating a TetR Transposon

Checked LBKan100 plates from yesterday. Made findings across these two days into a table below.

Strain	Growth on LBTet15	Growth on LBKan100
pTnMod-Okm	No	Yes
pEx18-Tc	Yes	No
pBE110	Yes	No

We transferred the tetracycline resistance gene from pEx18-Tc into pTnMod-Okm to create the pBE110. Our results so far confirm that the resistance of kanamycin was lost and the tetracycline resistance was gained in the pBE110.

Today, ran PCR to amplify the Kanamycin resistance gene using primers KanP forward/reverse. PCR parameters:

      1. 95C 5 minutes

      2. 54C 1 minute

      3. 72C 1 minutes

      4. 95C 10 seconds

      5. Repeat 2-4 steps 25 times

      6. 72C 5 minutes

      7. 20C infinitely

Ran PCR amplicons (Tet from yesterday, and Kan from today) on 1% gel using EZ Vision loading dye. 10 uL amplicon + 2 uL EZ vision.

Top lanes. Tetracycline amplicons.

Well 1	Well 2	Well 3	Well 4	Well 5	Well 6	Well 7
Hi Lo DNA Marker	pBE100	pBE110 A	pEx18-Tc (positive control)	pTnMod-Okm (neg control)	pBE110 B	Hi Lo DNA Marker

Bottom lanes. Kanamycin amplicons.

Well 1	Well 2	Well 3	Well 4	Well 5	Well 6	Well 7
Hi Lo DNA Marker	pBE100	pBE110 A	pEx18-Tc (negative control)	pTnMod-Okm (pos control)	pBE110 B	Hi Lo DNA Marker

Somewhat troubling results. The tetracycline resistance gene was identified in the Well 4 (top lanes), the pEx18-Tc, which is what was expected. pTnMod-Okm didn't amplify the tet resistance gene, which was also expected. However, it was only aplified in the pBE100 and not any of the pBE110 (A or B). However, both pBE110 A and B were able to grow on tetracycline. Maybe the tet resistance gene was altered during the ClaI/StuI digestion to create pBE110 and then the primers I used to amplify the tet res gene no longer amplified.

Kanamycin results (bottom lane) was also interesting. Only pTnMod-Okm was amplifed, which was hoped for. Neither pBE100 or any of the pBE110 had the Kanamycin gene, which was intended. 

So, the kanamycin resistance gene was not present in the pBE110, but neither was the tetracycline resistance gene. These results need some further interpreting.