Niklas Boess, Alexandra Cassano, John Connolly, Sanjukta DuXa ...
Undergraduate Category: Physical and Life Sciences Degree Level: BS Abstract ID#: 850
Variant Polymerase β mRNA Isola4on and Introduc4on to Biochemistry Techniques Niklas Boess, Alexandra Cassano, John Connolly, Sanjukta DuHa, Emma Fridley, Christopher Joshi Abstract
Students in the Honors Strauss Laboratory Directed Study have spent the spring semester learning basic techniques used in the biochemistry laboratory. These include: proper pipeHe use and calibraPon, PtraPons to form important biological buffers, sterile techniques, protein assays, preparaPon of soluPons, and proper creaPon and maintenance of scienPfic records. Students also submit detailed and professional laboratory reports every week to demonstrate criPcal thinking and a deep understanding of the theory behind their experiments. In addiPon, the students have learned the techniques necessary to isolate cDNA of zebrafish (Danio rerio) DNA polymerase ß (PolB), which will be further studied in the laboratory. These techniques include designing polymerase chain reacPon (PCR) primers, performing PCR and gel electrophoresis to examine PCR products, ligaPng the PCR products into an expression vector, transforming bacteria, and preparing minipreps. The PCR primers must be designed to isolate only the desired PolB sequence, and to effecPvely insert it into the plasmid. The Strauss laboratory first discovered that a variant of PolB is found in a 26 kilodalton form in D. rerio embryos. An analog of this protein has been found in mouse embryos. Furthermore, expressed sequence tags (ESTs) have been idenPfied in zebrafish, mouse and human for this PolB form. PolB is involved in the BER pathway, and usually has a mass of 39 kilodaltons. The cDNA encoding the 26 kilodalton form of the protein will eventually be used to create expression vectors for further study of the protein in early embryogenesis.
1 2 3 4 5 MW
130 95 70 43 34 26 15
Figure 1.
Samples of PtraPons performed and analyzed in the laboratory. From top-‐lea to boHom: Hepes and HCl, Hepes and NaOH, AcePc acid and C2H3NaO2. Red arrows indicate the pKa.
Ovalbumin Ac4n Bovine serum albumin Lane 1) Protein ladder 2) 10 mg/mL BSA 3) 5 mg/mL BSA 4) 1 mg/mL BSA 5) Zebrafish lysate
Figure 4.
Understanding the importance of pH in biological reac4ons: Titra4on curves and pKa
The above photo depicts an SDS-‐PAGE gel run with an unknown concentraPon of zebrafish lysate protein. Bovine serum albumin (BSA), which shares properPes with the zebrafish protein, was used as a comparison. From lea to right, the lanes contain a protein standard, 10 mg BSA, 5 mg BSA, 1 mg BSA, and 50 µg of zebrafish lysate. The lysate contains some idenPfiable proteins, such as gelsolin (90 kDa), ovalbumin (45 kDa), and acPn (42 kDa), all common in eukaryotes. However, these need to be confirmed by mass spec.
Understanding how pH and pKa work in relaPon to proteins and nucleic acids, students Ptrated various biologically important acids with HCl, NaOH and their conjugate bases (In this case, acetate.) The Henderson-‐Hasselbach equaPon was used with the PtraPon curves to find the pKa, the equivalence point and the half equivalence point.
Resolving proteins by SDS-‐ polyacrylamide gel electrophoresis (SDS-‐PAGE)
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis is a useful laboratory procedure used to separate proteins based on size. In SDS-‐PAGE a nega4vely charged detergent, SDS, coats and denatures proteins so that the proteins become negaPvely charged and linear. These proteins are loaded into a polyacrylamide gel, which is placed in a gel box with electric current running from top to boHom. The negaPvely-‐charged SDS-‐coated proteins are pulled through the gel towards the posi4ve pole. Small proteins move faster than large ones, so they can be separated by size. The gel is then stained with Coomassie blue, and analyzed by comparing unknown proteins to a protein standard. The members of Strauss lab ran an SDS-‐PAGE in order to compare and tentaPvely idenPfy the proteins in zebrafish lysate. Lab members esPmated the lengths of the proteins by comparing them to the molecular weights of the protein standards. With respect to the final project, SDS-‐ PAGE can be used to confirm the presence of shortened DNA PolB protein.
Applica4on of newly learned techniques to independent project: Clone the gene for variant PolB
Figure 2.
Gel electrophoresis of pME18s with RNF40 insert, restricted with HindIII and EcoRI. Lane 1) 1kb ladder 2,5) Uncut plasmid 3,6) HindIII digest 7) EcoRI digest 4,8) HindIII + EcoRI digest
Introduc4on
PreparaPon for lab research began last semester in BIOL 2299. In this class, students learned the basics of biological genePcs, with focus on the human genome. DNA replicaPon, transcripPon, translaPon, bacterial transformaPon, and other biotechnological techniques were explored, including: PCR, SDS-‐PAGE, Next Gen Sequencing, and Sanger Sequencing as well as ethical ramificaPons of the human genome project. The class placed a strong emphasis on searching primary literature databases such as PubMed as well as the gene and protein databases. Students also analyzed an unknown DNA sequence and examined the three dimensional structure of the protein that it encoded. Overall, the course introduced crucial biological understandings and fostered a spirit of scienPfic inquiry. This semester, the directed study has implemented many of the techniques covered last semester in class. Titra4ons were conducted using acids or bases common to biochemistry labs. Also, a series of restric4on digests were used to determine the size and locaPon of an insert in a pME18s plasmid. A Bradford assay, using BSA as a standard, was performed to determine the concentraPon of a zebrafish protein. E. coli bacteria were transformed and culPvated on agar plates before minipreps were performed to isolate the plasmid. Lastly, an SDS gel was used to determine the concentraPon of zebrafish protein in a given sample. Through these laboratory experiments, students learned the techniques necessary for complePng the final project, isolaPng the DNA PolB variant found in zebrafish embryos.
Gelsolin
The experiments performed thus far in the directed study will culminate in a final project aimed at finding a novel zebrafish gene. The Strauss lab had previously discovered a shorter variant of DNA PolB in zebrafish embryos. Using a cDNA library, complementary primers can be created that will anneal to the target gene, and extract it via PCR. Running a DNA digest will help confirm the idenPfy of the sequence. Eventually, the gene will be cloned into an expression vector for closer study of the protein. Aaer complePon of the final project, students will have training in the most common biochemistry laboratory techniques and be prepared to conduct more advanced research in academic or commercial labs.
Data already in hand (from Brian Dobosh)
Isola4on of variant PolB cDNA
As a final project for the course, students will use the skills and techniques learned throughout the semester to isolate cDNA of the 29 kilodalton Danio rerio PolB variant. This protein is involved in the BER pathway for DNA repair. The protein, along with its truncated form, is shown in Figure 6. The isolaPon will be accomplished by designing PCR primers analogous to both ends of the desired cDNA sequence. An important component of primer design is ensuring that the primers are complementary to restric4on sites that are on the plasmid, but are not in the gene. The cDNA will then be extracted using PCR to amplify only the desired sequence. Once the sequence has been isolated, gel electrophoresis will be used to confirm that the desired sequence was correctly isolated. This PolB cDNA will then be cloned into an expression vector for further study.
Insert
Figure 3.
Plasmid map of pME18s with RNF40 insert, with predicted locaPon of insert.
Figure 5: qRT PCR comparing transcripts from early and late embryos.
Plasmid
Resolving DNA fragments by agarose gel electrophoresis
In order to fully understand gel electrophoresis and its applicaPons, members of the lab were tasked with finding the length of an insert in pME18S plasmid. The reported length of the plasmid is 3392 base pairs (bp). Each member of the lab chose two restricPon endonucleases with which to esPmate the length of the insert. Aaer preparing the digest, lab members ran gel electrophoresis to quanPfy the length of DNA fragments produced. Based upon the results recorded, we found that the insert was roughly 3100 bp in length, and was located between the two XhoI sites on the plasmid (see Figure 3).
Graph of the PolB variants, shown with exons 1, 3, 4, 5, 13, and 14. This shows that the difference between the transcripts at 1.5 and 24 hpf at the 5’ end of the gene and the 3’ end of the gene.
Figure 6.
DNA PolB comparison of full-‐length protein sequence and truncated form found in early embryos.
References and Acknowledgements
Brunelle, J.L. and Green, L. Chapter twelve– one dimensional SDS-‐Polyacrylamide Gel Electrophoresis Methods in Enzymology 541, 151-‐159. ForPer, S., Yang, X., Bennet, R.A.O., Wang, Y. and Strauss, P.R. Base excision repair in early zebrafish development: evidence for DNA polymerase switching and standby AP nuclease acPvity Biochemistry 48, 5496-‐5405. Pei, D., Yang, X., Liu, W., Guikema, J.E.J, Schrader, C.E. and Strauss, P.R. A novel regulatory circuit in base excision repair involving AP endonuclease 1, Creb 1 and DNA polymerase β Nucleic Acids Res. 39, 3156-‐3165. Dobosh, Brian. PolB graph and protein with exons schema.
Each of us parPcipaPng in the Honors Directed Study would also like to thank Professor Phyllis Strauss for her dedicaPon to mentoring us throughout the semester and providing us with such an incredible opportunity.
Contact informa4on Niklas Boess: [email protected] Alexandra Cassano: [email protected] John Connolly: [email protected] Sanjukta DuHa: [email protected] Emma Fridley: [email protected] Christopher Joshi: [email protected]
Professor Phyllis Strauss: [email protected] 617-‐373-‐3492 106 Lake Hall
Variant Polymerase β mRNA Isola4on and Introduc4on to Biochemistry Techniques Niklas Boess, Alexandra Cassano, John Connolly, Sanjukta DuHa, Emma Fridley, Christopher Joshi Abstract
Students in the Honors Strauss Laboratory Directed Study have spent the spring semester learning basic techniques used in the biochemistry laboratory. These include: proper pipeHe use and calibraPon, PtraPons to form important biological buffers, sterile techniques, protein assays, preparaPon of soluPons, and proper creaPon and maintenance of scienPfic records. Students also submit detailed and professional laboratory reports every week to demonstrate criPcal thinking and a deep understanding of the theory behind their experiments. In addiPon, the students have learned the techniques necessary to isolate cDNA of zebrafish (Danio rerio) DNA polymerase ß (PolB), which will be further studied in the laboratory. These techniques include designing polymerase chain reacPon (PCR) primers, performing PCR and gel electrophoresis to examine PCR products, ligaPng the PCR products into an expression vector, transforming bacteria, and preparing minipreps. The PCR primers must be designed to isolate only the desired PolB sequence, and to effecPvely insert it into the plasmid. The Strauss laboratory first discovered that a variant of PolB is found in a 26 kilodalton form in D. rerio embryos. An analog of this protein has been found in mouse embryos. Furthermore, expressed sequence tags (ESTs) have been idenPfied in zebrafish, mouse and human for this PolB form. PolB is involved in the BER pathway, and usually has a mass of 39 kilodaltons. The cDNA encoding the 26 kilodalton form of the protein will eventually be used to create expression vectors for further study of the protein in early embryogenesis.
1 2 3 4 5 MW
130 95 70 43 34 26 15
Figure 1.
Samples of PtraPons performed and analyzed in the laboratory. From top-‐lea to boHom: Hepes and HCl, Hepes and NaOH, AcePc acid and C2H3NaO2. Red arrows indicate the pKa.
Ovalbumin Ac4n Bovine serum albumin Lane 1) Protein ladder 2) 10 mg/mL BSA 3) 5 mg/mL BSA 4) 1 mg/mL BSA 5) Zebrafish lysate
Figure 4.
Understanding the importance of pH in biological reac4ons: Titra4on curves and pKa
The above photo depicts an SDS-‐PAGE gel run with an unknown concentraPon of zebrafish lysate protein. Bovine serum albumin (BSA), which shares properPes with the zebrafish protein, was used as a comparison. From lea to right, the lanes contain a protein standard, 10 mg BSA, 5 mg BSA, 1 mg BSA, and 50 µg of zebrafish lysate. The lysate contains some idenPfiable proteins, such as gelsolin (90 kDa), ovalbumin (45 kDa), and acPn (42 kDa), all common in eukaryotes. However, these need to be confirmed by mass spec.
Understanding how pH and pKa work in relaPon to proteins and nucleic acids, students Ptrated various biologically important acids with HCl, NaOH and their conjugate bases (In this case, acetate.) The Henderson-‐Hasselbach equaPon was used with the PtraPon curves to find the pKa, the equivalence point and the half equivalence point.
Resolving proteins by SDS-‐ polyacrylamide gel electrophoresis (SDS-‐PAGE)
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis is a useful laboratory procedure used to separate proteins based on size. In SDS-‐PAGE a nega4vely charged detergent, SDS, coats and denatures proteins so that the proteins become negaPvely charged and linear. These proteins are loaded into a polyacrylamide gel, which is placed in a gel box with electric current running from top to boHom. The negaPvely-‐charged SDS-‐coated proteins are pulled through the gel towards the posi4ve pole. Small proteins move faster than large ones, so they can be separated by size. The gel is then stained with Coomassie blue, and analyzed by comparing unknown proteins to a protein standard. The members of Strauss lab ran an SDS-‐PAGE in order to compare and tentaPvely idenPfy the proteins in zebrafish lysate. Lab members esPmated the lengths of the proteins by comparing them to the molecular weights of the protein standards. With respect to the final project, SDS-‐ PAGE can be used to confirm the presence of shortened DNA PolB protein.
Applica4on of newly learned techniques to independent project: Clone the gene for variant PolB
Figure 2.
Gel electrophoresis of pME18s with RNF40 insert, restricted with HindIII and EcoRI. Lane 1) 1kb ladder 2,5) Uncut plasmid 3,6) HindIII digest 7) EcoRI digest 4,8) HindIII + EcoRI digest
Introduc4on
PreparaPon for lab research began last semester in BIOL 2299. In this class, students learned the basics of biological genePcs, with focus on the human genome. DNA replicaPon, transcripPon, translaPon, bacterial transformaPon, and other biotechnological techniques were explored, including: PCR, SDS-‐PAGE, Next Gen Sequencing, and Sanger Sequencing as well as ethical ramificaPons of the human genome project. The class placed a strong emphasis on searching primary literature databases such as PubMed as well as the gene and protein databases. Students also analyzed an unknown DNA sequence and examined the three dimensional structure of the protein that it encoded. Overall, the course introduced crucial biological understandings and fostered a spirit of scienPfic inquiry. This semester, the directed study has implemented many of the techniques covered last semester in class. Titra4ons were conducted using acids or bases common to biochemistry labs. Also, a series of restric4on digests were used to determine the size and locaPon of an insert in a pME18s plasmid. A Bradford assay, using BSA as a standard, was performed to determine the concentraPon of a zebrafish protein. E. coli bacteria were transformed and culPvated on agar plates before minipreps were performed to isolate the plasmid. Lastly, an SDS gel was used to determine the concentraPon of zebrafish protein in a given sample. Through these laboratory experiments, students learned the techniques necessary for complePng the final project, isolaPng the DNA PolB variant found in zebrafish embryos.
Gelsolin
The experiments performed thus far in the directed study will culminate in a final project aimed at finding a novel zebrafish gene. The Strauss lab had previously discovered a shorter variant of DNA PolB in zebrafish embryos. Using a cDNA library, complementary primers can be created that will anneal to the target gene, and extract it via PCR. Running a DNA digest will help confirm the idenPfy of the sequence. Eventually, the gene will be cloned into an expression vector for closer study of the protein. Aaer complePon of the final project, students will have training in the most common biochemistry laboratory techniques and be prepared to conduct more advanced research in academic or commercial labs.
Data already in hand (from Brian Dobosh)
Isola4on of variant PolB cDNA
As a final project for the course, students will use the skills and techniques learned throughout the semester to isolate cDNA of the 29 kilodalton Danio rerio PolB variant. This protein is involved in the BER pathway for DNA repair. The protein, along with its truncated form, is shown in Figure 6. The isolaPon will be accomplished by designing PCR primers analogous to both ends of the desired cDNA sequence. An important component of primer design is ensuring that the primers are complementary to restric4on sites that are on the plasmid, but are not in the gene. The cDNA will then be extracted using PCR to amplify only the desired sequence. Once the sequence has been isolated, gel electrophoresis will be used to confirm that the desired sequence was correctly isolated. This PolB cDNA will then be cloned into an expression vector for further study.
Insert
Figure 3.
Plasmid map of pME18s with RNF40 insert, with predicted locaPon of insert.
Figure 5: qRT PCR comparing transcripts from early and late embryos.
Plasmid
Resolving DNA fragments by agarose gel electrophoresis
In order to fully understand gel electrophoresis and its applicaPons, members of the lab were tasked with finding the length of an insert in pME18S plasmid. The reported length of the plasmid is 3392 base pairs (bp). Each member of the lab chose two restricPon endonucleases with which to esPmate the length of the insert. Aaer preparing the digest, lab members ran gel electrophoresis to quanPfy the length of DNA fragments produced. Based upon the results recorded, we found that the insert was roughly 3100 bp in length, and was located between the two XhoI sites on the plasmid (see Figure 3).
Graph of the PolB variants, shown with exons 1, 3, 4, 5, 13, and 14. This shows that the difference between the transcripts at 1.5 and 24 hpf at the 5’ end of the gene and the 3’ end of the gene.
Figure 6.
DNA PolB comparison of full-‐length protein sequence and truncated form found in early embryos.
References and Acknowledgements
Brunelle, J.L. and Green, L. Chapter twelve– one dimensional SDS-‐Polyacrylamide Gel Electrophoresis Methods in Enzymology 541, 151-‐159. ForPer, S., Yang, X., Bennet, R.A.O., Wang, Y. and Strauss, P.R. Base excision repair in early zebrafish development: evidence for DNA polymerase switching and standby AP nuclease acPvity Biochemistry 48, 5496-‐5405. Pei, D., Yang, X., Liu, W., Guikema, J.E.J, Schrader, C.E. and Strauss, P.R. A novel regulatory circuit in base excision repair involving AP endonuclease 1, Creb 1 and DNA polymerase β Nucleic Acids Res. 39, 3156-‐3165. Dobosh, Brian. PolB graph and protein with exons schema.
Each of us parPcipaPng in the Honors Directed Study would also like to thank Professor Phyllis Strauss for her dedicaPon to mentoring us throughout the semester and providing us with such an incredible opportunity.
Contact informa4on Niklas Boess: [email protected] Alexandra Cassano: [email protected] John Connolly: [email protected] Sanjukta DuHa: [email protected] Emma Fridley: [email protected] Christopher Joshi: [email protected]
Professor Phyllis Strauss: [email protected] 617-‐373-‐3492 106 Lake Hall
