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Glucose susceptibility

Submitted by cnwokemodoih on Tue, 04/23/2019 - 22:03

Glucose growth condition is typically a form of osmotic stress. When a gene, SFAR4, was knocked out in Arabidopsis, the mutant plant was susceptible to glucose osmotic conditions and had lower germination rates than overexpression transgenic lines and wild-type. Under mannitol osmotic stress conditions, germination rates in mutant plants were not significantly lower than those in overexpression transgenic lines and wild-type. This indicates that the susceptibility to glucose is not due to osmotic stress but due to the mutated gene.

Lab 4

Submitted by cnwokemodoih on Mon, 04/22/2019 - 18:59

To get a basic idea of the Bradi3g27407.2 gene expression pattern, we used the e-FP browser. This web-based tool gave us a graphical summary of our gene expression data. We analyzed the pictogram, chart and table outputs and noted the tissues and organs where our gene is expressed. We explored more about our gene expression pattern using the PlaNet gene expression clustering program. In addition, we retrieved gene expression data from Phytozome for different Brachypodium distachyon plant growth conditions. We made figures for select conditions.

Novel GDSL-type esterase

Submitted by cnwokemodoih on Sat, 04/20/2019 - 00:07

A novel GDSL-type esterase, SFAR4 was found in Arabidopsis thaliana. In this study, knockout mutants and overexpression lines were established. The absence and overexpression of the SFAR4 gene had no effect on germination rate when compared with the wild-types. However, in the presence of glucose, the lines with overexpressed SFAR4 had significantly higher germination rates than wild-type and knockout mutants. In contrast, the presence of mannitol did not affect germination rates. This finding suggests that perhaps SFAR4, a GDSL-type esterase plays a role in glucose susceptibility and glucose metabolic pathway regulation in germinating seeds. In this same study, the expression of fatty acid metabolic genes are shown to be differential in SFAR4 overexpression lines and knockout mutants. SFAR4 overexpression lines show increases levels of metabolism pathway components while knockout mutant lines are accompanied by lower levels of fatty acid metabolism gene expression. This indicates that SFAR4 plays a role in fatty acid metabolism.

 

BS1

Submitted by cnwokemodoih on Thu, 04/18/2019 - 09:14

Another study done on plants gives a different dimension to GDSL esterase activity . In this study, a forward genetic screen reveals a brittle leaf sheath 1 (bs1) mutant that is characterized by brittle leaf sheaths. Further genomic investigation, reveals that the BS1 gene codes for a GDSL esterase that removes acetyl moieties from xylan backbones. Because xylan is a major component of secondary cell walls, secondary cell wall architecture is compromised in bs1 mutants. This suggests that this GDSL esterase allows proper secondary wall patterning to occur. BS1 is ubiquitously expressed in plants but more in vascular tissues. Subcellular examination revealed localization in the Golgi apparatus. This is interesting because prior to this discovery, no GDSL esterase/lipase had been discovered to have polysaccharide esterase activity.

 

GDSL Lipases/esterases

Submitted by cnwokemodoih on Wed, 04/17/2019 - 15:39

The idea of GDSL-lipases/esterases as modulators of immunity is relevant not just in Oryza sativa but also Arabidopsis and hot pepper (Gao et al., 2017). As such, it won’t be out of place to predict that our protein is involved in the modulation of immunity. The plant and subcellular localization patterns are also interesting because they suggest that the same might be the case for our protein. However, we can not draw conclusively on where our protein is expressed; it might be different.

 

SFAR4

Submitted by cnwokemodoih on Mon, 04/15/2019 - 22:08

A novel GDSL-type esterase, SFAR4 was found in Arabidopsis thaliana. In this study, knockout mutants and overexpression lines were established. The absence and overexpression of the SFAR4 gene had no effect on germination rate when compared with the wild-types. However, in the presence of glucose, the lines with overexpressed SFAR4 had significantly higher germination rates than wild-type and knockout mutants. In contrast, the presence of mannitol did not affect germination rates. This finding suggests that perhaps SFAR4, a GDSL-type esterase plays a role in glucose susceptibility and glucose metabolic pathway regulation in germinating seeds. In this same study, the expression of fatty acid metabolic genes are shown to be differential in SFAR4 overexpression lines and knockout mutants. SFAR4 overexpression lines show increases levels of metabolism pathway components while knockout mutant lines are accompanied by lower levels of fatty acid metabolism gene expression. This indicates that SFAR4 plays a role in fatty acid metabolism.

 

Chemical transformation of One Shot Top 10 E.Coli cells

Submitted by cnwokemodoih on Wed, 04/10/2019 - 09:54

Chemical transformation of E. Coli cells is the process by which bacterial cell asre made to take up desired plasmids. Chemical transformation is performed after TOPO cloning. Some preparatory steps include equilibrating the water bath to 420C, warming selective plates at 720C for 30 minutes and thawing vials of One Shot Top 10 E.Coli cells on ice. To start off, 2 microliters of TOPO cloning reaction products are added into vials, mixed gently and then incubated on ice for 5-30 minutes. The cells are heat shocked for 30 second at 420C without shaking before being placed on ice for 2 minutes. 250 microliters of S.O.C. medium is added, the tube is capped tightly and then shook horizontally at 200RPM and 370C for 1 hour. 

Aim of Research Project

Submitted by cnwokemodoih on Tue, 04/09/2019 - 22:34

The aim of the project is to gain more insight into the function of the alpha-4 GABAA receptor by the deep examination of subunit expression in the brain and spinal cord of zebrafish. The in-situ hybridization procedure done previously (Monesson-Olson et al., 2018), allowed the visualization of six subunit expression patterns, including that of alpha-4. This gave a rough picture of subunit function. Since the mutation screen revealed interesting alpha-4 subunit activity, further analysis is required. Conducting this fluorescent in-situ hybridization procedure will allow me to identify not just where the alpha-4 subunit is expressed but also what structures it interacts with. Knowing what the alpha-4 subunit interacts with will aid in predicting its function. By extension, this will provide a framework for developing treatments that can suppress the effects of related neurological disorders like epilepsy.

 

Topocloning PP

Submitted by cnwokemodoih on Fri, 04/05/2019 - 12:47

Topocloning is a faster and more efficient version of the traditional cloning method. Cloning is the process by which a DNA sequence of interest is integrated into a vector plasmid. This is done traditionally by cutting the plasmid with a restriction enzyme and then ligating either end of the sequence to the open ends of the plasmid. The ligation step is performed by an enzyme called ligase. The use of two different enzymes in two different steps makes the traditional cloning method ineffective. Many errors occur using this method. The advent of topocloning has circumvented the use of two enzymes and reduced errors, by the use of a single enzyme called topoisomerase 1. The vector comes open with two toposiomerase 1 enzymes on either end of the open vector. Once the sequence of interest comes into the gap, the topoisomerase 1 enzymes catalyze the ligation step, closing up the vector and integrating the appropriate sequence into the vector.

Topocloning

Submitted by cnwokemodoih on Fri, 04/05/2019 - 12:45

Topocloning is a faster and more efficient version of  traditional cloning. Cloning is the process by which a DNA sequence of interest is integrated into a vector plasmid. This is done traditionally by cutting the plasmid with a restriction enzyme and then ligating either end of the sequence to the open ends of the plasmid. The ligation step is typically performed by an enzyme called ligase. The use of two different enzymes in two different steps makes the traditional cloning method ineffective. Many errors occur using this method. The advent of topocloning has circumvented the use of two enzymes, by the use of a single enzyme called topoisomerase 1. The vector comes open with two toposiomerase 1 enzymes on either end of the open vector. Once the sequence of interest comes into the gap, the topoisomerase 1 enzymes catalyze the ligation step, closing up the vector and integrating the appropriate sequence into the vector.

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