How to do colony PCR with Agrobacterium tumefaciens (or other difficult to lyse cells)

If the cells don't lyse well, they result in a smear instead of a band.

Example 1% agarose gel of Agrobacterium tumefaciens colony PCR:

Lanes:

1. Colony PCR with pre-boiled Agrobacterium produces the expected band size.
2. DNA ladder, NEB 2-log
3. Colony PCR with unboiled Agrobacterium added directly from the LB agar plate fails, results in streaks. This happens with the standard colony PCR protocol for E. coli with a 10 minute step at 95C. It's not enough to lyse Agrobacterium, so the PCR fails.

Publication: Tight regulation of plant immune responses by combining promoter and suicide exon elements

Tania L. Gonzalez, Yan Liang, Bao N. Nguyen, Brian J. Staskawicz, Dominique Loqué, and Ming C. Hammond. Nucleic Acids Research 2015 Aug 18; 43(14): 7152–7161. doi: 10.1093/nar/gkv655

Links

• PubMed ID: 26138488
• PubMed Central ID: PMC4538838
• Nucleic Acids Research journal article
• Sequences
  
• HyP5SM cassette (intron-exon-intron) sequence
• OsL5 sequence - OsL5 promotes HyP5SM exon skipping

HyP5SM model

• Default retention of the "suicide exon" HyP5SM results in a non-productive mRNA.
• Co-expression of OsL5 promotes protein expression of the gene of interest by altering HyP5SM splicing.
• All parts of the HyP5SM gene expression system come from plant sequences.
• The HyP5SM system is expected to function in all dicot plants.
  

What is HyP5SM and how does it regulate genes?

HyP5SM is an inducible gene regulation method for dicot plants. This method works by cloning in a DNA sequence (a splicing cassette made with hybrid monocot and dicot sequences) into your gene of interest. It is very flexible, can be inserted at various possible sites, and is not sensitive to nearby sequence. The splicing is controlled by a monocot (rice) splice factor which does not bind to the endogenous dicot homolog.

The "off" version of the RNA is degraded by nonsense mediated decay, a natural cleanup process that removes mRNAs with stop codons in the middle. This is why HyP5SM is called a "suicide exon", also sometimes referred to as a "poison exon". It is not dangerous to humans. It just results in targeted RNA breakdown by the plant, so the plant does not make the protein. HyP5SM works so well that it is able to regulate proteins that trigger plant immune responses (very sensitive phenotypes). This low background is the key benefit of HyP5SM and the reason why researchers might want to use it over inducible promoters. Inducible promoters are notoriously "leaky", showing measurable levels of protein even without induction. HyP5SM removes this background.

The "on" version of the RNA results in complete removal of the splicing cassette, and thus a protein which has no sequence alterations. Because the splicing cassette is completely removed, HyP5SM is a "traceless" gene induction system.

The HyP5SM inserted sequence is 100% of plant origin, a hybrid of endogenous Arabidopsis thaliana (dicot) and Oryza sativa (rice, monocot) P5SM sequences. There is no bacterial or other non-plant sequence required. 

I tested the sensitivity of the gene regulation by cloning HyP5SM into plant pathogen proteins to see if I could regulate the plant's effector-triggered immune responses, but the pathogen proteins are absolutely not required. They were just my genes of interest. You can use your own.