I am transforming Agrobacterium (AGL-1) with large multi-gene constructs (around 20kb). I have tried to minimise homologous sequences but I do have some similar sequences within the plasmid. When I do colony PCRs directly from the agro colonies I see the correct band sizes. However, when I grow up the colonies into culture, do a miniprep and do the same PCRs, some rearrangements seem to have occurred such that some of the plasmid appears intact but certain areas won't PCR at all. Is there any way to limit recombination in agro, ie growing conditions, strain, etc?
1. Use of Agrobacterium strain with RecA genotype should be avoided. Reports have shown that RecA has promoted the homologous recombination in E. Coli and Agrobacterium (see attachments #1 and #2).
2. The rearrangement of your plasmid DNA in Agrobacterium can be caused by RecA-depended mechanism or RecA-independent mechanism. Some research results indicated that RecA-independent mechanism is the main rout for Agrobacterium (attachment #1), while some indicated that RecA involved in rearrangement (deletion) in Agrobacterium (attachment #2).
3. However, look like that the RecA gene of AGL1 Agrobacterium strain you are using is mutated (see attachments #2 and #3), so the RecA-independent mechanism could be the cause for your DNA rearrangements (but double-check your AGL1 to see if it is 'recA' mutant).
u can prevent homologous recombination by using a negative selection marker in your plasmid that you use. pGKO2 is a good vector for homologous recombination constructs. it has thyidine kinase as negative selection marker, that will prevent growth of cells if integrated to the genome. also thymidine kinase will get integrated to genome via homologous recombination only if the recombination has occurred at a site other than the target site. so u can use it for screening colonies as well.
PRI201 a takara vector with kanamycin selection marker can also be a good option,
other option is redo directional cloing with diffrent restriction sites in MCS
1. Use of Agrobacterium strain with RecA genotype should be avoided. Reports have shown that RecA has promoted the homologous recombination in E. Coli and Agrobacterium (see attachments #1 and #2).
2. The rearrangement of your plasmid DNA in Agrobacterium can be caused by RecA-depended mechanism or RecA-independent mechanism. Some research results indicated that RecA-independent mechanism is the main rout for Agrobacterium (attachment #1), while some indicated that RecA involved in rearrangement (deletion) in Agrobacterium (attachment #2).
3. However, look like that the RecA gene of AGL1 Agrobacterium strain you are using is mutated (see attachments #2 and #3), so the RecA-independent mechanism could be the cause for your DNA rearrangements (but double-check your AGL1 to see if it is 'recA' mutant).
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