About
12
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833
Citations
Introduction
Cis or trans. That's what matters.
Additional affiliations
January 2019 - September 2021
November 2021 - January 2024
January 2014 - April 2014
Position
- Student internship
Description
- Study of involvement of ROCK1 in glycosylation of enzymes, particularly AtCKX2.
Education
September 2010 - January 2018
Publications
Publications (12)
Cytokinins are plant hormones, derivatives of adenine with a side chain at the N6‑position. They are involved in many physiological processes. While the metabolism of trans‑zeatin and isopentenyladenine, which are considered to be highly active cytokinins, has been extensively studied, there are others with less obvious functions, such as cis‑zeati...
Cytokinins (CKs) are a class of phytohormones affecting many aspects of plant growth and development. In the complex process of CK homeostasis in plants, N-glucosylation represents one of the essential metabolic pathways. Its products, CK N7- and N9-glucosides, have been largely overlooked in the past as irreversible and inactive CK products lackin...
Main conclusion
Five poplar CHASE-containing histidine kinase receptors bind cytokinins and display kinase activities. Both endogenous isoprenoid and aromatic cytokinins bind to the receptors in live cell assays.
Abstract
Cytokinins are phytohormones that play key roles in various developmental processes in plants. The poplar species Populus × can...
Main conclusion
Isoprenoid and aromatic cytokinins occur in poplar as free compounds and constituents of tRNA, poplar isopentenyltransferases are involved in the production of isoprenoid cytokinins, while biosynthesis of their aromatic counterparts remains unsolved.
Cytokinins are phytohormones with a fundamental role in the regulation of plant gro...
Degradation of the plant hormone cytokinin is controlled by cytokinin oxidase/dehydrogenase (CKX) enzymes. The molecular and cellular behavior of these proteins is still largely unknown. In this study, we show that CKX1 is a type-II single-pass membrane protein that predominantly localizes to the endoplasmic reticulum (ER). This indicates that this...
Almost 25 years ago, an enzyme named zeatin cis–trans isomerase from common bean has been described by Bassil et al. (1993). The partially purified enzyme required an external addition of FAD and dithiothreitol for the conversion of cis-zeatin to its trans- isomer that occurred only under light. Although an existence of this important enzyme involv...
Cytokinins (CKs) are an important group of phytohormones. Their tightly regulated and balanced levels are essential for proper cell division and plant organ development. Here we report precise quantification of CK metabolites and other phytohormones in maize reproductive organs in the course of pollination and kernel maturation. A novel enzymatic a...
Cytokinin hormones are important regulators of development and environmental responses of plants that execute their action
via the molecular machinery of signal perception and transduction. The limiting step of the whole process is the availability
of the hormone in suitable concentrations in the right place and at the right time to interact with t...
The catabolism of cytokinins is a vital component of hormonal regulation, contributing to the control of active forms of cytokinins and their cellular distribution. The enzyme catalyzing the irreversible cleavage of N(6)-side chains from cytokinins is a flavoprotein classified as cytokinin dehydrogenase (CKX, EC 1.5.99.12). CKXs also show low cytok...
Plant hormones, cytokinins (CKs), have been for a long time considered to be involved in plant responses to stress. However, their exact roles in processes linked to stress signalization and acclimatization to adverse environmental conditions are unknown. In this study, expression profiles of the entire gene families of CK biosynthetic and degradat...
Questions
Questions (124)
I'm doing recetly plenty of cloning and to find E. coli colonies with my desired construct, I do colony PCR.
However, as I am preparing brand new constructs, using new primers for the colony PCR, I do touch-down PCR, as I cannot optimize the conditions beforehand and I have no positive control, because I take it, that it should provide sufficiently non-stringent conditions to get some amplification and at the same time increased specificity in comparison to simple PCR.
But my PI says that touchdown PCR is not appropriate for colony PCR. At first I disregarded it, but then I thought about it more. It's true that it should increase specificity if there is something to amplify, but if it's not, it will probably allow non-specific amplification (which happens in our case - that's why I'm asking).
However, how else to do it? As I wrote, usually I have no positive control and often I use even several pairs of primers. Of course I always check the annealing temperature at NEB's Tm calculator (as we use their polymerases) and I don't go that much lower, usually 1-2°C. So even if I were running normal PCR, the non-specific amplification would be probably there, right?
Hello everyone,
We are currently doing some deletions in one of our plasmids and after obtaining colonies, we are trying to do colony PCR, but it gives us just some smear (incl. positive control). Considering that the marker looks good, it's likely problem with the PCR, rather than the electrophoresis.
At first, colleague did the PCR, her gel is on the picture named original. She got at least some resemblence of bands. Then I did new colony PCR and it's even worse.
My setup of the PCR was as follows:
5x OneTaq Standard Reaction Buffer 4 ul
10 mM dNTPs 0.4 ul
10 uM fw primer 0.4 ul
10 uM rev primer 0.4 ul
boiled bacteria 2.5 ul
OneTaq DNA Pol 0.4 ul
water 11.9 ul
This polymerase was brand new, we got it few weeks back, nobody used it yet. I used higher concentration of the polymerase, as they recommend for longer amplicons.
I did touch-down PCR, because the primers are not tested yet. The cycling was as follows:
94°C 30 s
94°C 20 s
54-0.5°C 30 s
68°C 10 min 10 x
94°C 20 s
49°C 30 s
68°C 10 min 30x
68°C 5 min
The elongation is so long, because I used the original plasmid as positive control, which should give amplicon of 7 kb with one pair of the primers. The other combinations of primers and templates should give amplicons between 1 kb and 4.5 kb.
I did two sets of primers, one is on the top row of the picture, the other is at the lower row of the picture. The positive control (which is isolated plasmid, not bacteria with the plasmid) is in the last well on top row and first well on lower row.
Any idea what could have gone wrong? Or should I try to change?
My plan at this moment is to try just with the positive control plasmid with my Taq polymerase, which I know works to check the primers. Otherwise I don't know.
My PI wants to use expression plasmid for E. coli with two cloning sites. We have pColA-Duet-1 and pACYC-Duet, but in those are both of the sites inducible by IPTG (if I understood it correctly). Are there any plasmids that would have two different inducible promoters?
