Navigation Links
Using XL1-Red Mutator Strain to Generate Esterase Variants

2

In Figure 2, plasmids isolated from E. coli colonies produced during mutation cycles using XL1-Red competent cells were transferred into a non-mutator E. coli host, then plated and incubated on LB/Amp agar plates. From these master plates, colonies were replica plated onto minimal-media agar containing rhamnose (to induce esterase production), substrates 1 or 2, and indicators (crystal violet and neutral red). Upon hydrolysis of substrates 1 or 2, the pH in the microenvironment of the colonies decreased and red spots are formed (from the indicator).

We achieved a good compromise between fast bacterial growth and reliable detection of positive variants by using plates with minimal media. From approximately 750 colonies, several putative mutant clones were identified based on the red color that developed on the agar plates after the colonies were incubated for 2 to 6 days at 37C.

The plasmids were isolated using colonies from the master plates and then transformed back into a non-mutator host. After the colonies were cultivated for 3 hours and induced with rhamnose for 4 hours, the esterase produced was isolated by sonification and subjected to preparative biotransformation. When the esterases capable of hydrolysing the 3-hydroxy ester 1 were sequenced, one variant contained two point mutations (A209D and L181V). In the indicator assay, the clone that produced this variant also gave the strongest red color and largest colony size.

The esterase gene is 843 bp long, and the double mutant was isolated after three mutation cycles. Each cycle is approximately 30 generations and the spontaneous mutation rate should yield approximately 1 mutation per 2,000 base pairs every 30 generations. This PFE mutant was subjected to another mutation cycle using XL1-Red competent cells, and approximately 9500
'"/>

Source:


Page: All 1 2 3 4 5

Related biology technology :

1. Low Abundance cDNA Cloned Using Stratagenes Human Universal cDNA Library
2. Oil-Free PCR Using the Hot Top Assembly
3. Functional Cloning Using ViraPort Retroviral cDNA Expression Libraries
4. Signal Transduction Reporting Systems Using Cis-Acting Enhancer Elements
5. Detect Released Oligosaccharides Using the Eagle Eye II Still Video Imaging System
6. Comparison of Growth Techniques and Media for the Purpose of Plasmid Isolation from E. coli Using the Eppendorf Perfectprep Plasmid Mini Kit
7. Genomic DNA Extraction from Buccal Swabs Using the Perfect gDNA Blood Mini Kit
8. Genomic DNA Extraction from Buffy Coat Using the Perfect gDNA Blood Mini Kit
9. Isolation of Genomic DNA from Saliva Using the Perfect gDNA Blood Mini Kit
10. Protocol for RNA Isolation Using TRIzol Reagent with Phase Lock Gel-Heavy
11. Genomic DNA Extraction from Buccal Swabs Using the Perfect gDNA Blood Mini Kit
Post Your Comments:
*Name:
*Comment:
*Email:
TAG: Using Red Mutator Strain Generate Esterase Variants

(Date:7/22/2014)... 22, 2014 The Board of Directors of BD ... declared a quarterly dividend of 54.5 cents per common share ... September 9, 2014. The indicated annual dividend rate is $2.18 ... is a leading medical technology company that partners with customers ... and evolving health needs. Our innovative solutions are focused on ...
(Date:7/22/2014)... within a phylum so full of mean little creatures, the ... its cruelty -- at least to crickets. Native to the ... most predatory sort of parasite. It swoops onto the back ... and leaves its wicked brood to invade, kill and consume ... would be possible without the fly,s ability to find a ...
(Date:7/22/2014)... Institute of Standards and Technology (NIST) and California ... design for an atomic clock that is based ... , The microcomb clock, featured on the cover ... Optica ,* is the first demonstration of ... conversion of optical frequencies to lower microwave frequencies. ...
(Date:7/22/2014)... in water with ultrasound and they,ll spin around their ... yet knows exactly. But researchers at the National Institute ... it,s fast. At up to 150,000 revolutions per minute, ... object submerged in liquid ever reported. , The discovery ... that they could be used not only for moving ...
Breaking Biology Technology:Fly-inspired sound detector 2Fly-inspired sound detector 3Fly-inspired sound detector 4'Comb on a chip' powers new NIST/Caltech atomic clock design 2NIST shows ultrasonically propelled nanorods spin dizzyingly fast 2
... Get Full-length cDNA , ... to improve production of full-length cDNA. This engineered reverse transcriptase, , ... href="http://www.ambion.com/catalog/CatNum.php?2048">ArrayScript , ... cDNA than wild type RTs, and rivals even ...
... Screening with small interfering RNAs , ... in biological pathways. This article analyzes ... of two strategies--transfection of individual , ... screening experiments. The increased effectiveness of testing , ...
... Isolate total RNA and protein from cultured cells or tissues , ... RNA and protein from cultured , cells , ... Ambion's new PARIS Kit uses a fast, simple procedure based ... native protein , from the same experimental sample. The kit ...
Cached Biology Technology:ArrayScript RT: Maximize Yields of Full-length cDNA 2ArrayScript RT: Maximize Yields of Full-length cDNA 3Are siRNA Pools Smart? 2Are siRNA Pools Smart? 3Are siRNA Pools Smart? 4Are siRNA Pools Smart? 5Are siRNA Pools Smart? 6Are siRNA Pools Smart? 7Are siRNA Pools Smart? 8Are siRNA Pools Smart? 9A Better Way to Isolate RNA AND Protein From the Same Sample 2A Better Way to Isolate RNA AND Protein From the Same Sample 3A Better Way to Isolate RNA AND Protein From the Same Sample 4A Better Way to Isolate RNA AND Protein From the Same Sample 5
(Date:7/22/2014)... July 14, 2014, by lightning from a weather system that ... four fires: Stokes Fire, Gold Hikes Fire, French Creek Fire ... are now one larger fire. The fires are burning in ... 243,000 acres in size. The present fire situation continues ... and firefighter safety is the priority on the Carlton Complex ...
(Date:7/22/2014)... MIAMI A new study that examined the survival ... as,unintentional bycatch in commercial longline fishing operations found ... with bigeye thresher, dusky, and scalloped hammerhead being ... the University of Miami (UM) Rosenstiel School of,Marine ... Ecosystem Science and Policy, provides,new information to consider ...
(Date:7/22/2014)... known as the ,1000 Bull Genomes Project, aims to ... cattle while also improving animal health and welfare. Results ... the whole genomes of 234 individual bulls whose direct ... reported in the journal Nature Genetics . , ... information to reduce or eliminate hereditary diseases and to ...
Breaking Biology News(10 mins):New study reveals vulnerability of sharks as collateral damage in commercial fishing 2Whole-genome sequencing of bulls in key beef and dairy breeds 2
... study comparing human walking and running motions and whether the ... these motions researchers at North Carolina State University show that ... the ankles generate more of the power when humans run. Knees ... The research could help inform ...
... Spain have used forensic mitochondrial DNA species identification techniques ... makes it possible to genetically differentiate between the fish, ... it easier to monitor the degree to which fisheries ... cell organelles is ideal for distinguishing between ...
... Beginning in June 2011, Springer will launch a ... Sciences ( HCIS ). As part of the ... Korea-based Future Technology Research Association International (FTRA). ... the official international journals of the FTRA. The journal ...
Cached Biology News:Hips take walking in stride; ankles put best foot forward in run 2Sardines and horse mackerel identified using forensic techniques 2Springer launches new open access journal with Korean research society 2
Borosilicate Glass. Certified for use with the Beacon 2000 Systems, 1,000 tubes/case...
... Engineered with new technology incorporating ... for unsurpassed flexibility, the new Varian ... advanced spectrometer available today., ,The Varian ... each transmitter and receiver channel, providing ...
Luminometers...
... Dr. P Set from different ... More organs maybe available upon ... two items from the whole set, ... set price. All Dr. P ...
Biology Products: