Hi all,
I am establishing a method to test the transformability of bacterial communities. I did the following:
1) I isolated gDNA from the community and marked them with a transposon which introduces two ME sequences.
2) I transformed this DNA to the same community
3) after incubation for a short while I centrifuged the cells, performed a DNAse I digestion on the pellet
4) I did a colony PCR on this pellet with primers binding to the transposon
The PCR worked, I got products with a length of ~500-1000 bp. I sequenced the PCR products with NGS to get the bacteria.
Well, DNAseI digestion should prevent false positives. I think even after washing, the DNA could bind extracellularly to the bacterial cells. (although the cell surface is negatively charged).
A PCR on non-marked metagenomic DNA showed a smear with only very small fragments < 100 bp. I excluded small reads of the sequencing data.
I am going to perform a control at which I will add a plasmid to non-competent E. coli. After DNAse treatment, I will perform a PCR to ensure that the DNAse digestion was successful.
Do you have any further ideas how I could prevent false positive transformants?
Any further ideas for a similar experiment?
Thanks!
Sonja Vanderhaeghen
Use sterile tools and labware, media, and reagents where appropriate or required in the workflow.
9. Make sure spreading rods and/or glass beads are sterile, and cell plating is performed under aseptic conditions
1. Check the genotype of the host strain for antibiotic resistance. For example, BL21 Star(DH3)pLysS cells are resistant to chloramphenicol, OmniMAX 2 T1R cells are resistant to tetracycline, and TOP10 cells are resistant to streptomycin.
2. Include a negative control without DNA in the transformation step for verification of antibiotic effectiveness. Assuming the correct genotype, untransformed cells should not survive the antibiotic selection.
Vector plasmid recombined into the host chromosome
3. The vector plasmid may have integrated into the bacterial chromosome, conferring antibiotic resistance. When this occurs, colonies may appear to lack the transforming DNA, since independently replicating plasmids cannot be detected by the usual screening methods (which selectively purify or detect the plasmids and not the chromosomal DNA). Use competent cells with the recA mutation to prevent recombination and allow stable propagation of the transforming plasmid vector.
For Satellite colonies
4. Limit the incubation time to
Use sterile tools and labware, media, and reagents where appropriate or required in the workflow.
9. Make sure spreading rods and/or glass beads are sterile, and cell plating is performed under aseptic conditions
Thanks, Maurice. Those are very good tips for a general cloning procedure!
However, I am transforming enrichment cultures, meaning a community. By selecting on antibiotics, I would loose too many bacteria! I am establishing a protocol to test natural communities for their transformability.
- Badges
- Science method