United States Department of Agriculture
Question
Asked 4th Jan, 2014
How can one know whether an antibiotic resistance gene in a plasmid could be expressed in a certain strain or not? Can alignment work?
As we know, an antibiotic resistance gene may be expressed in E coli, but not in agrobacteria. If we could predict the expression ability of a certain antibiotic resistance gene through the alignment of the recognition (cis-acting elements)?
Most recent answer
I did more reading. Your plasmid seems to be OK unless something unexpected happened during cloning. Op den Camp et al. (2011) Plant Physiology 157:2013-2022 (www.plantphysiol.org/cgi/doi/10.1104/pp.111.187526) used a similar vector. You might also want to take a look at this other vector: Untergasser A, Bijl GJM, Liu W, Bisseling T, Schaart JG, et al. (2012) One-Step Agrobacterium Mediated Transformation of Eight Genes Essential for Rhizobium Symbiotic Signaling Using the Novel Binary Vector System pHUGE. PLoS ONE 7(10): e47885. doi:10.1371/journal.pone.0047885. Dr. Jan G. Schaart is on ResearchGate- he may be able to help you better.
All Answers (12)
Zagazig University
This can be done through studying the promoter region :. Take a look at the attached article to see how we modify any palsmid structure and expression
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Smt. K. W. College Sangli 416416. Maharashtra. INDIA
Well you may try, but personally I have my reservations. The only possibility could be that the plasmid did not recombine with the parent chromosome and hence did not express. Could you be isolate the plasmid intact from Agrobacterium sp? If No, then it could have been digested by the endonuclease of that organism.
United States Department of Agriculture
I worked with gene expression in Agrobacterium in graduate school a few decades ago so I may be a little rusty. You did not give much detail, but I think the problem may be that your plasmid cannot replicate in Agrobacterium. It can take quite a variety of plasmids (unfortunately most are low-copy number plasmids), but not ColE1-like, which is the base of most cloning/expression vectors.
Huainan Normal University
To Jai Ghosh,
Did the genes on the plasmid express only when it was recombined with the parent chromosome? Why is That? Couldn't the plasmid gene express solely?
To Chin-Yi Chen,
I agree with the low copy number plasmid assumption. But, do you approve, in the mean time, that the antibotic resistent genes should also express in Agrobacterium?
United States Department of Agriculture
Unless it requires specific positive regulators or sigma factors that are not present in Agrobacterium, almost any gene should be able to express, although the efficiency may not be optimal. I remembered using carbenicillin, chlorampphenicol, kanamycin, spectinomycin and tetracycline for selection, some at much lower concentrations (I can't remember the exact genes though). You misunderstood me regarding the low copy plasmids- I meant that most Agrobacterium plasmids used in the expression studies are low-copy and maybe difficult to work with when you do the cloning in E. coli. What exactly is the incompatibility group of your plasmid?
Huainan Normal University
To Chin-Yi Chen,
I am using a gateway destination plasmid (figure provided) to RNAi an interested gene. The plasmid contained kanaR, CmR and specR genes, but I can't sure which should be used after the transformation into Agrobacteria A4, which has RifR gene. Can you give me some suggestions?
Huainan Normal University
According to the previous experiments, when this plasmid was transfered into Ecoli (DH5alfa), Ecoli showed SmR and no KanaR character.
I followed the references related to this plasmid application and found that KanaR gene was designed for transformed plant selection. But I couldn't find the plant promoter driving this KanaR gene. And what's the purpose of placing CmR in the middle of the introns?
United States Department of Agriculture
I am not familiar with this plasmid. The map did not label the replication origin it is using. I did a quick search and I think this is a "regular" gateway plasmid. If I am correct, it will not replicate in Agrobacterium, no matte what antibiotics you tried. Did the reference actually tell you that it could be used in Agrobacterium? You need to find Agrobacterium-specific vectors with engineered T-DNA region and appropriate replication origin. (I assume that you are doing RNAi in plant.) Try this site for background information: http://www.ndsu.edu/pubweb/~mcclean/plsc731/transgenic/transgenic3.htm
Huainan Normal University
To Chin-Yi Chen,
Thank you so much. I will learn more about the characters of this plasmid. After I figure it out, I'll let you know.
United States Department of Agriculture
I did more reading. Your plasmid seems to be OK unless something unexpected happened during cloning. Op den Camp et al. (2011) Plant Physiology 157:2013-2022 (www.plantphysiol.org/cgi/doi/10.1104/pp.111.187526) used a similar vector. You might also want to take a look at this other vector: Untergasser A, Bijl GJM, Liu W, Bisseling T, Schaart JG, et al. (2012) One-Step Agrobacterium Mediated Transformation of Eight Genes Essential for Rhizobium Symbiotic Signaling Using the Novel Binary Vector System pHUGE. PLoS ONE 7(10): e47885. doi:10.1371/journal.pone.0047885. Dr. Jan G. Schaart is on ResearchGate- he may be able to help you better.
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