Creating a TetR Transposon

Goal: The purpose of this study is to create a transposon with a tetracycline antibiotic marker. We have a pTnMod-Okm in stock, but we are unable to use it for our purposes because of the kanamycin marker. We would like perform random transposon mutagenesis on a number of Stenotrophomonas maltophilia isolates, but we can't use the pTnMod-Okm because most of the isolates are already resistant to kanamycin. So, we would like to replace the kanamycin marker with a tetracycline marker.

Previously: We amplified the Tetracycline resistance gene from the knockout vector pEx18Tc (from Link) using tetracycline amplifying primers. The tetracycline primers were created using the Geneious software with a NotI flanking region on the forward primer and StuI on the reverse primer. NotI and StuI are restriction enzyme sites that are also located on the the pTnMod-Okm outside the kanamycin gene. We had previously digested the pTnMod-Okm with StuI + NotI and ligated in the amplified tetracycline gene using T4 ligase. The ligated plasmid was transformed into electrically competent DH5a. The created plasmid was called "pBE100", however it did not fully replace the kanamycin resistance gene as it was still resistant. We believe that is because the orientation of the kanamycin gene was not what we previously believed and we used pTnMod-Okm' and not pTnMod-Okm. So, this study is to cut out the remaining kanamycin resistance gene in pBE100, ligate the plasmid back together, and then transform it into chemically competent DH5a.

On Friday, May 16, 2014:

1. Performed plasmid prep of pBE100 using Zyppy kit (Link).

2. Performed double restriction digest on pBE100 using ClaI and StuI to excise the kanamycin gene.
          10 uL of plasmid DNA
          5 uL of 10x Cutsmart Buffer
          1 uL of StuI enzyme
          1 uL of ClaI enzyme
          33 uL of ddH2O
     Incubate at 37 C heat block (with water in it) for 30 minutes.

3. Performed DNA clean up kit on the digested plasmid using DNA Clean & Concentrator (Link).

4. Performed Mung Bean Nuclease digest (New England BioLabs).
          17 uL of purified digested plasmid DNA in elution buffer
          2 uL of 10x NEBuffer 2
          1 uL of Mung Bean Nuclease enzyme
    Incubate at 30 C heat block (with water in it) for 30 minutes

5. Performed DNA clean up kit using DNA Clean & Concentrator (Link).

6. Visualized DNA on a FlashGel DNA system (Link). Removed FlashGel from packaging, attached to rig, and filled wells with ddH2O. Loaded 4 uL of DNA combined with 1 uL of FlashGel Loading Dye (total of 5 uL into each well). Used FlashGel DNA Marker. Ran gel at 275 volts for 5 minutes.

Well 1	Well 2	Well 3	Well 4	Well 5	Well 6	Well 7	Well 8	Well 9	Well 10	Well 11
Flash Gel Marker	ClaI + StuI digested pBE100	ClaI + StuI digested pBE100	purified  ClaI + StuI digested pBE100	purified ClaI + StuI digested pBE100	purified Mung Bean Digested pBE100	purifiedMung Bean Digested pBE100	pEx18 Tc	pEx18 Tc:: GW	pTn Mod-Okm	Flash Gel Marker

7. Performed Quick Ligation Protocol (New England BioLabs).
          10 uL purified Mung Bean Digested plasmid DNA
          10 uL 2x Quick Ligation Buffer
          1 uL of Quick T4 DNA Ligase
    Incubate at room temperature for 5 minutes. Chilled on ice after until use.
    Newly created plasmid (formally pBE100) is now called "pBE110".

8. Performed DNA clean up kit using DNA Clean & Concentrator (Link).

9. Transformed into One Shot OmniMax 2 T1 Chemically Competent Cells (invitrogen) according to the manufacturer's protocol. Used pUC19 as a control. Also, transformed pEx18Tc plasmid DNA (running low on stock)

10. Diluted each transformant 1:50 in LB broth. Plated pBE110 onto LB Tet15 and pUC19 onto LB Amp100. Also plated pEx18Tc onto LB Tet15. Incubated at 37C for 48 hours.

On May 18, 2014

11. Brothed isolated colonies into 10 mL of LB with appropriate antibiotic. Incubated at 37 C, shaking at 225 rpm.
        Note: protect tet plates from light by wrapping in aluminum foil.


//EWW