Williams A- Poster Presentation (1)
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TEMPLATE DESIGN © 2008 www.PosterPresentations.com Inserting Wild Type Calreticulin into the pRSET-A Vector Ashia S Williams, Lystranne Maynard PhD, Akintunde Akinyemi MS. Department of Chemistry, Howard University, Washington, DC 20001 Goal The Purpose In this study, ampicilin resistant mCRT was cloned in order to understand the acetylation mechanism of CRT outside the ER. To achieve this, wild type CRT DNA was removed from a pGEM T-Easy vector and placed into a the more useful pRSET-A vector. The Significance Calreticulin (CRT) is a conserved multifaceted protein localized in the endoplasmic reticulum (ER) whose roles inside the ER include calcium regulation and protein folding inside cells. It has been shown that acetylation, a post- translational modification, is the mechanism by which CRT fulfills some of these roles within the ER. Previous studies have localized CRT in the endoplasmic reticulum however, recent studies have found traces of this protein in liver, brain and other cellular regions. Background Information Methods Instrumentation •Centrifuge –DNA mini prep •260nm UV/Vis Absorption of DNA –260nm absorption of DNA –260/280 is used to check for purity –mCRT 99.1ng/uL –pRSET-A 83.2ng/uL •Incubator and Incubator Shaker –Transformation and Overnights •37 degree water bath –digestion Developing Protocol Initially, the calreticulin DNA was cloned out of a pGEX vector and attempted to be inserted directed into pRSET-A vector using Ncol and BamH1 enzymes in a simultaneous digestion. However, due to steric hindrance, mCRT was unable to be inserted into the vector. Next, HindIII and BamH1 primers were used to introduce these endonuclease sites to the ends of the mCRT in pGEX and then insert the mCRT into a T-vector in a sequential digestion. This step was successfully completed. It was concluded that inserting mCRT into a T-vector would make it easier to extract the mCRT from the T-vector and then insert it into a pRSET-A vector. . Results Gel pRSET-A Small is the mCRT Large T-vector Excise 160mg mCRT 100mg pRSET-A Medical Application Human Health and Disease Conclusion Acknowledgement Absence of CRT is embryonically lethal Presence of non-ER extracellular CRT Rheumatoid Arthritis Inflammatory bowel disease Alzheimer Disease Upon insertion of mCRT into the pRSET-A vector a resistance to ampicilin is inherited therefore a successful insertion will be indicated by the growth of colonies in a LB-AMP environment. Further Investigation The resulting DNA can be further confirmed via DNA sequencing. To develop a computational model of a novel receptor, a controlled study will be performed on wild type and mutant CRT to examine the role acetylation may play in the exit of CRT from the ER Discovery of Calreticulin (CRT) •1974 Skeletal Muscle cells •Thomas Ostwald and David MacLennan •Conserved protein that assist in protein chaperoning and calcium regulation in the endoplasmic reticulum (ER). –A network of membranous tubules within the cytoplasm of a eukaryotic cell •Other names –HABP, Calregulin OPTIONAL LOGO HERE OPTIONAL LOGO HERE Structure pRSET- A T- vector mCRT DNA ladder Lystranne Maynard, PhD Akintunde Akinyemi, MS Ronald E. McNair Program Howard University-Funding Department of Biology Department of Chemistry Winston Anderson PhD, Biology
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Transcript of Williams A- Poster Presentation (1)
TEMPLATE DESIGN © 2008
www.PosterPresentations.com
Inserting Wild Type Calreticulin into the pRSET-A VectorAshia S Williams, Lystranne Maynard PhD, Akintunde Akinyemi MS.Department of Chemistry, Howard University, Washington, DC 20001
Goal
The Purpose
In this study, ampicilin resistant mCRT was cloned in order to understand the acetylation mechanism of CRT outside the ER. To achieve this, wild type CRT DNA was removed from a pGEM T-Easy vector and placed into a the more useful pRSET-A vector.
The Significance
Calreticulin (CRT) is a conserved multifaceted protein localized in the endoplasmic reticulum (ER) whose roles inside the ER include calcium regulation and protein folding inside cells. It has been shown that acetylation, a post-translational modification, is the mechanism by which CRT fulfills some of these roles within the ER. Previous studies have localized CRT in the endoplasmic reticulum however, recent studies have found traces of this protein in liver, brain and other cellular regions.
Background Information
Methods
Instrumentation
•Centrifuge –DNA mini prep
•260nm UV/Vis Absorption of DNA–260nm absorption of DNA–260/280 is used to check for purity –mCRT 99.1ng/uL–pRSET-A 83.2ng/uL
•Incubator and Incubator Shaker–Transformation and Overnights
•37 degree water bath–digestion
Developing Protocol
Initially, the calreticulin DNA was cloned out of a pGEX vector and attempted to be inserted directed into pRSET-A vector using Ncol and BamH1 enzymes in a simultaneous digestion. However, due to steric hindrance, mCRT was unable to be inserted into the vector.
Next, HindIII and BamH1 primers were used to introduce these endonuclease sites to the ends of the mCRT in pGEX and then insert the mCRT into a T-vector in a sequential digestion. This step was successfully completed.
It was concluded that inserting mCRT into a T-vector would make it easier to extract the mCRT from the T-vector and then insert it into a pRSET-A vector..
Results
GelpRSET-ASmall is the mCRTLarge T-vector
Excise160mg mCRT100mg pRSET-A
Medical Application Human Health and Disease
Conclusion
Acknowledgement
Absence of CRT is embryonically lethal
Presence of non-ER extracellular CRTRheumatoid ArthritisInflammatory bowel diseaseAlzheimer Disease
Upon insertion of mCRT into the pRSET-A vector a resistance to ampicilin is inherited therefore a successful insertion will be indicated by the growth of colonies in a LB-AMP environment.
Further InvestigationThe resulting DNA can be further confirmed via DNA sequencing. To develop a computational model of a novel receptor, a controlled study will be performed on wild type and mutant CRT to examine the role acetylation may play in the exit of CRT from the ER
Discovery of Calreticulin (CRT)•1974 Skeletal Muscle cells•Thomas Ostwald and David MacLennan•Conserved protein that assist in protein chaperoning and calcium regulation in the endoplasmic reticulum (ER).–A network of membranous tubules within the cytoplasm of a eukaryotic cell
•Other names–HABP, Calregulin
OPTIONALLOGO HERE
OPTIONALLOGO HERE
Structure
pRSET-A
T-vector
mCRT
DNA ladder
Lystranne Maynard, PhDAkintunde Akinyemi, MSRonald E. McNair ProgramHoward University-FundingDepartment of BiologyDepartment of ChemistryWinston Anderson PhD, Biology
