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.