Actually I'm transforming the AGL1 strain with pGKO2 containing our gene construct, but i don't know the reason why the Agrobacterium can't grown after electroporation with the plasmid? what can be the possible reason? the electrocompetency of the AGL1 is compromised or there is any problem in the gene construct? The question that comes in my mind is that if the vector doesn't have the gene construct and it goes into the AGL1, can it grow on the medium with antibiotics? thanks in advance
Hello Latif,
If this is your pGKO2 (https://www.addgene.org/63617/, although there is also a Gateway version), it does have kan resistance and if you propagate the backbone the cells should be able to grow in kan.
You have a few possible causes for your problem:
- the vector is not what you think it is. Instead of transforming agrobacterium, propagate your clone in E. coli first (you should obtain kan-resistant colonies). This is highly advisable since the efficiency of cloning is usually low; if you try to transform agro directly the transformation efficiency will be too low to have a chance to see a transformed cell. If you do not get kan-resistant coli colonies, your plasmid is something else (as long as those cells work with other plasmids, of course).
- if E. coli grows but agro fails, it could be that the latter are no longer competent, or that you are killing them during the electroporation. The easiest way to test this to take a plasmid that has been previously transformed into agro to assess competency.
As regards your question, whether you can be transforming the plasmid without insert, just take a look at the plasmid map and revise your cloning procedure. Are you excising the kan resistance gene at any point?
From your question, I would say you just need to transform E. coli first to propagate the plasmid, then check it by restriction digestion to ensure that everything looks like it should (bands of expected sizes), and then electroporate agro.
For plasmid map drawing I'd recommend the free version of Snapgene (http://www.snapgene.com/).
Cheers,
Ruben
pGKO2 has a negative selection marker thymidine kinase..meaning if empty vecctor is transformed cells wont grow. kindly refer to the paper of Khang et al 2005 where they decrbibe properties of pGKO1 and pGKO2. it is a gateway cloning vector meant for creating knockouts using homologous recombination.
@Sansrity. I agree, the vector should be checked. However if the vector is the addgene one, it is noted that it confers kan resistance and that the propagation strain is DH5alpha, so I assumed the thymidine kinase construct would not affect the bacteria.
Good coment,
Ruben
@Ruben Alvarez-Fernandez thank you for your valuable comments.. In fact i propagate the plasmid first in E.coli in LB medium containing Kan. and then i isolate the plasmid by using a plasmid extraction kit. the extracted plasmid is then transferred to the AGL1 agrobacterium by electroporation. but no growth is observed. Now i have to check the competency of the agrobacterium cells by transforming it with another vector.
@Sansrity Sinha... I know HSVtk is used as a negative selection marker, but the selection system work in fungus just to differentiate ectopic transformants from the right transformants... (ectopic transformants express both the negative and positive selection markers, while transformants resulting from gene replacement lack the negative selection marker. Therefore, the negative selection should eliminate ectopic transformants , facilitating the identication of transformants resulting from gene replacement since they are resistant to this negative selection)
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