Working with TRBO vectors.

The following are a few comments regarding questions you may have regarding TRBO vectors.

1. To grow up DNA for cloning:

A. Transform plasmid into E. coli (example DH5a).

B. Plate on LB plates with 50 ug/ml kanamycin for selection.

C. Grow liquid cultures in LB with 50 ug/ml Kan.

It is probably best to do a large scale prep (eg 500 mls culture), because plasmid is fairly low copy.

2. For cloning:

Digest pJL TRBO DNA with appropriate enzymes.

The multiple cloning site of TRBO has PacI-AvrII and NotI sites.

Note that PacI and NotI are both 8 base cutters.

AvrII has sticky ends compatable with AvrII, NheI, SpeI and XbaI.

Gel isolate Vector after digestions and treatment with alkaline phosphatase. can help (but is often not necessary).

IF GEL ISOLATING EITHER VECTOR OR INSERT…. DO NOT expose DNAs to UV!

Instead use “Clare Dark reader” and Syber Gold (Invitrogen/molecular probes) to visualize DNAs in gel. This is really really important.

Use zymogen DNA isolation kit to isolate DNAs from gel. Lets you elute DNAs in small volume. Also elute DNAs in 10 mM Tris pH 8.0 (NO EDTA).

When setting up ligations: Use a 2 or 3 fold MOLAR excess of insert to vector. DO NOT EXCEED 3 FOLD MOLAR EXCESS OF INSERT!! THIS IS ALSO REALLY IMPORTANT.

Estimate [] of Vector and insert DNA samples using Nanodrop UV/Vis spectrophotometer.

Set up ligations with 50 ng of vector (in 10 or 20 ul final volume).

Use Invitrogen 5X ligation buffer (has PEG in it).

  • AVOID multiple (ex more than 5) freeze-thaws of this buffer. Also make sure buffer is dissolved--there can sometimes be a precipitate.
  • Aliquot buffer into small volumes (25 ul or so), store at -20C. Throw out aliquot after using 5 freeze thaws.

Ligate overnight at 16C.

Transform 1 ul of ligation into 50 ul of DH5a competent cells (high efficiency-- > 108 CFU/ug DNA)

I usually purchase high effic DH5a cells (from Invitrogen or Bioline). When they arrive, thaw cells, aliquot into 50ul samples in sterile, chilled 1.5 ml tubes.

3. For Agroinfection:

A. pJL TRBO is based on the minibinary vector pCB301 which has T-DNA borders, but no vir genes, or genes needed for conjugation into other bacteria. Transform pJL TRBO based plasmid into Agro containing a ‘helper plasmid’ that can provide the factors (vir genes) needed for T-DNA processing and transfer.

Transform Agro with your method of choice. (note: conjugation/tri-parental mating will not work with pJL TRBO).

B. Plate transformed Agro on LB plates with 50 ug/ml Kanamycin, and the appropriate antibiotics for the selection of the helper plasmid.

For example if transforming into Agro GV3101 plate on LB with 50 ug/ml kanamycin, 25 ug/ml gentamycin and 10 ug/ml rifampicin.

C. Grow Agro colonies in LB with 50 ug/ml kanamycin, 25 ug/ml gentamycin and 10 ug/ml rifampicin for overnight to 24 hours. Culture at ~ 28C, 250 rpm.

i. Dilute overnight agro culture 1:10 into fresh LB Kan50/Gent25 with 20 uM Acetosyringone.

Grow at ~ 28 C, 250 rpm to OD600 of ~ 1.0 (should take 4-5 hours).

ii.. Spin down OD600 Agro culture.

Resuspend in equal volume of Agro-induction media.

Agro-induction media:

10 mM MES pH 5.7

10 mM MgCl2

100 uM acetosyringone.

iii. Let Agro sit in agroinduction media for 2 to 24 hours at room temp.

iv. Infiltration:

If desired, dilute “induced” agro culture from the OD 1.0 with

10 mM MES pH 5.7, 10 mM MgCl2 solution.

Pull up into 1ml syringe with no needle.

Infiltrate into underside of leaves.

v. Observe plants under UV to see GFP expression from TRBO-G control.

Expect to begin seeing faint GFP by about 2 ½ days post infiltration.

TRBO vector moves cell-to–cell but not systemically.

With GFP reporter expression seems to plateau between 4 and 6 days post infiltration.

4. For more information or for referencing TRBO vector in publications or talks please use:

Lindbo JA. (2007) TRBO: A High Efficiency Tobacco Mosaic Virus RNA Based Overexpression Vector. Plant Physiol.

5. In case you are interested in sequencing your insert once it is cloned into TRBO, the following sequence is the sequence of TRBO ca. 150 nts upstream and downstream of the PacI-AvrII-NotI cloning sites (in bold). Use this sequence to design primers for sequencing if desired.

GAACAAGAACTATAGAAATGTTAAGGATTTTGGAGGAATG

AGTTTTAAAAAGAATAATTTAATCGATGATGATTCGGAGG

CTACTGTCGCCGAATCGGATTCGTTTTAAATAGATCTTAC

AGTATCACTACTCCATCTCAGTTCGTGTTCTTGTCAttaa

ttaacggcctagggcggccgcGGTCCTGCAACTTGAggta

gtcaagatgcataataaataacggattgtgtccgtaatca

cacgtggtgcgtacgataacgcatagtgtttttccctcca

cttaaatcgaagggttgtgtcttggatcgcgcgggtcaaa