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Group b

  1. 1. Cloning Simulation Project 2003 GTB 204 - Molecular Biology Techniques
  2. 3. Abstract <ul><li> In this cloning project, we have chosen the erythropoietin gene to clone. We get the gene sequence from the gene bank. After this, we go through all the routine procedures in cloning project, such as searching the gene sequence, looking for its ORF, restriction sites, primers as well as searching for an appropriate vector to clone it. We also compare the gene with BLAST and multiple DNA alignment. </li></ul><ul><li> From the information and data we get, we organize the needed steps to get our gene of interest. First, we do the PCR to amplify our gene sequence and TA Cloning before doing the insertion into the vector and transformation into the host cell. Then, we do all the necessary screening to verify and confirm the correct specific cloned gene. Finally, we do the expression and extraction and the purification of the protein to obtain the pure protein that we want. </li></ul>
  3. 4. Introduction <ul><li>Erythropoietin, (EPO), a glycoprotein hormone produced primarily by cells of the peritubular capillary endothelium of the kidney, is responsible for the regulation of red blood cell production. </li></ul><ul><li>Secondary amounts of the hormone are synthesized in liver hepatocytes of healthy adults. In premature as well as full term infants, the liver is the primary site of EPO production. </li></ul><ul><li>The kidney becomes the primary site of EPO synthesis shortly after birth. EPO production is stimulated by reduced oxygen content in the renal arterial circulation. </li></ul><ul><li>Circulating EPO binds to EPO receptors on the surface of erythroid progenitors resulting in replication and maturation to functional erythrocytes by an incompletely understood mechanism. </li></ul>
  4. 5. <ul><li>Among the haematopoietic growth factors, erythropoietin is one of few that behaves like a hormone. It is unique because its production, under normal circumstances, is controlled solely at the level of its gene, by tissue hypoxia and not by the absolute number of circulating erythrocytes. Hypoxia is the sole physiologic stimulus for erythropoietin production, and an excess of oxygen suppresses its production but never completely. </li></ul><ul><li>The gene encoding human erythropoietin was cloned in 1985 leading to the production of recombinant human EPO (rhu-EPO). Rhu-EPO has been used successfully in a variety of clinical situations to increase production of red blood cells. </li></ul><ul><li>Erythropoietin may be given intravenously or more effective subcutaneously (doses: 50- 300 units/ kg) </li></ul><ul><li>Essential for end- stage renal disease and also used for anemia of chronic disorders such as AIDS, cancer and aplastic anemia </li></ul>
  5. 6. Objective <ul><ul><ul><li>To know the processes in doing cloning simulation project so as to understand the concept of molecular biology and biotechnology. </li></ul></ul></ul><ul><ul><ul><li>To obtain the experiences necessary in cloning strategy. </li></ul></ul></ul><ul><ul><ul><li>To choose/select the gene we want to clone from Genbank. </li></ul></ul></ul><ul><ul><ul><li>To search for the sequence of the erythropoietin gene. </li></ul></ul></ul><ul><ul><ul><li>To analyze the Epo gene for its ORF, restriction enzyme site, amino acid sequence, and molecular weight. </li></ul></ul></ul><ul><ul><ul><li>To design primer for PCR amplification and clone screening. </li></ul></ul></ul><ul><ul><ul><li>To choose the appropriate expression vector. </li></ul></ul></ul><ul><ul><ul><li>To design the cloning strategy – PCR cloning or cloning on a restriction site. </li></ul></ul></ul><ul><ul><ul><li>To select the appropriate clone screening method. </li></ul></ul></ul><ul><ul><ul><li>To choose the appropriate purification method to purify recombinant protein. </li></ul></ul></ul><ul><ul><ul><li>To explore the use of the protein and its commercial value. </li></ul></ul></ul>
  6. 9. Step1: Hunting for the gene <ul><li>Hunting for a gene is the primary and tough step in the cloning simulation project. We have tried to search for the data of the gene we wanted, that is erythropoietin gene from Genbank at the website . </li></ul><ul><li>NCBI is a national resource center that gathers useful molecular biology information in the field of biotechnology. It is established in 1988 and it remains active in conducting research in computational biology, developing powerful software tools for analyzing genome data, creating public databases and disseminating biomedical information for a better understanding of molecular process regarding human health and diseases. </li></ul><ul><li>After searching, we successfully obtained the gene sequence that codes for erythropoietin. Its accession number is NM_005232. </li></ul><ul><li>It provides the complete coding sequence (cds), as shown below. </li></ul>
  7. 12. Step 2: Determining Amino Acid Sequence & Molecular Weight of Erythropoietin Gene
  8. 17. <ul><li>It is known that one amino acid is coded by one codon. For example, Methionine, ATG is equal to 3 base pairs. </li></ul><ul><li>Also, one amino acid has a molecular weight of 110 Dalton. </li></ul><ul><li>Hence, the molecular weight of erythropoietin can be estimated approximately by multiplying the total number of amino acids with 110 Dalton, as below: </li></ul><ul><li>Molecular weight for erythropoietin gene = (total base pairs / 3) </li></ul><ul><li>X 110 Dalton </li></ul><ul><li> = (2930/3) X 110 Dalton </li></ul><ul><li> = 107433.333 Dalton </li></ul><ul><li> = 107.43 kD </li></ul>
  9. 18. Step 3: Looking for Open Reading Frame (ORF) <ul><li>Open Reading Frame (ORF) is a DNA sequence that contains a series of codon, which can be translated into protein. It is characterized by long stretches of codon that code for protein/polypeptide in the same reading frame that begins with a start codon (ATG or GTG) and ends with a stop codon (TGA, TAG or TAA). </li></ul><ul><li>For the erythropoietin gene, the ORF can be defined by accessing to the website . The ORF Finder is a graphical analysis tool which finds all open reading frames of a selectable minimum size in a user’s sequence or in a sequence already in the database. This tool identifies all open reading frames using the standard or alternative genetic codes. This analysis is to make sure that the region of gene of interest that is from 88 - 3018 bp can be expressed. </li></ul><ul><li>From this analysis, we have found that: </li></ul><ul><li>1) The gene sequence of interest starts with a start codon (ATG) and ends with a stop codon (TAG). </li></ul><ul><li>2) Below is the result of the analysis: </li></ul>
  10. 19. ORF Finder (Open Reading Frame Finder)
  11. 21. Step 4: Looking for the Restriction Enzyme Site <ul><li>Restriction enzyme will cut the gene sequence to give fragment of DNA. Different restriction enzyme has different restriction sites on DNA strand. Restriction enzyme analysis had to be done to determine whether there is any restriction endonuclease enzymes that cut inside the sequence of the gene of interest. Only the restriction site for the endonucleases that do not cut inside the sequence of the gene of interest are chosen when designing primers for PCR. </li></ul><ul><li>We accessed the website, to analyze the restriction enzyme site in the sequence. We copied the gene sequence from G enbank and pasted it into the particular blank and performed the analysis. </li></ul><ul><li>However, in our project, we do not use restriction enzyme because we are using the method of TA Cloning which will be discussed later. </li></ul><ul><li>Result: </li></ul>
  12. 24. Step 5: Comparing the Gene Sequence with other similar Gene Sequences by BLAST Analysis and Multiple DNA Sequence Alignment <ul><li>After verifying the open reading frame and restriction enzyme of erythropoietin gene, comparison of the gene sequence with other similar gene sequences available in the gene bank is done by using the BLAST analysis and multiple DNA sequence alignment. </li></ul>
  13. 25. BLAST (Basic Local Alignment Search Tool) <ul><ul><ul><li>The request ID of our finding for the analysis through the Gene Bank website is 1061360686-27838-1190898.BLASTQ3. </li></ul></ul></ul>
  15. 32. Multiple DNA Sequence Alignment (CLUSTALW) <ul><li>We have done the analysis of multiple DNA sequence alignment according to the results we got from the BLAST analysis. We copied the gene sequence of Homo sapiens EPO, Mus musculus EPO and Rattus norvegicus similar to EPO and then did the analysis through . </li></ul><ul><li>An alignment of three or more sequences with gaps inserted in the sequences such that residues with common structural positions and/or ancestral residues are aligned in the same column. Clustal W is one of the most widely used multiple sequence alignment programs </li></ul><ul><li>The results are as follow: </li></ul>
  16. 40. Step 6: Designing primer for PCR Amplification and screening <ul><li>Primer is a short nucleic acid containing free 3’ hydroxyl group that forms base pairs with a complementary template strand and functions as the starting point for additional nucleotides to copy the template. For PCR amplification, two primers are needed, the forward primer and reverse primer. We designed the primers manually as it is more accurate. The reverse primer designed contains the complementary base pair for 6 His tag and enterokinase site which is essential for protein purification. </li></ul><ul><li>Forward primer </li></ul><ul><li>5’ atg gag cgg cgc tgg ccc 3’ </li></ul><ul><li>Reverse primer </li></ul><ul><li>5’ tca gtg gtg gtg gtg gtg gtg gat gac gat gac aag gtc ctt gaa tcc ctg 3’ </li></ul><ul><li>Footnote: </li></ul><ul><li>> Complementary base pairs for erythropoietin coding sequence </li></ul><ul><li>> Complementary base pairs for 6 His tag coding sequence </li></ul><ul><li>> Complementary base pairs for enterokinase site coding sequence </li></ul>
  17. 41. Calculation of the Annealing Temperature <ul><li>There is a formula to calculate the annealing temperature, which requires the sequence of the primer because the amounts of specific nucleotides are needed. The formula is as follow: </li></ul><ul><li>Melting Temperature (Tm) = (A+T)2 + (G+C) 4 </li></ul><ul><li>Tm for forward primer = (2+2)2 + (8+6)4 = 64 ºC </li></ul><ul><li>Tm for reverse primer = (2+5)2 + (3+5)4 = 46 ºC </li></ul><ul><li>Annealing Temperature (Ta) = Tm - (2-5 ºC) </li></ul><ul><li>Ta for forward primer = 64 ºC – 2 = 6 2 ºC </li></ul><ul><li>Ta for reverse primer = 4 6 ºC – 2 = 4 4 ºC </li></ul><ul><li>Using the formula above, the melting temperature for the forward primer is 64 ºC and reverse primer is 4 6 ºC . By comparing the annealing temperature for forward and reverse primer, the lowest temperature (ºC) was chosen. Therefore, the annealing temperature is 44 ºC . </li></ul>
  18. 42. Step 7: Designing the Vector <ul><ul><ul><li>Description on the vector </li></ul></ul></ul><ul><li>The pCR®4-TOPO vector has 3´-T overhangs for cloning Taq -amplified PCR products. The vector is supplied linearized and topoisomerase Iactivated. Some of their convenient features include: </li></ul><ul><li>a) T7 promoter/priming sites for sequencing and </li></ul><ul><li>in vitro transcription/translation </li></ul><ul><li>b) Eco R I sites flanking the PCR product insertion site for easy removal of inserts </li></ul><ul><li>c) Kanamycin and ampicillin resistance genes for choice of selection in E. coli </li></ul>
  19. 43. Step 8: PCR <ul><li>Principle </li></ul><ul><li>Our next step is doing the Polymerase Chain Reaction (PCR), a technique that is used to amplify specific region of DNA, in order to produce enough DNA for further investigation. The number of nucleotides in the DNA strand are variable depending on the gene. PCR method comes in 3 main steps: </li></ul><ul><li>i. Denaturation process where the DNA was heated to denature the DNA paired strands into two. </li></ul><ul><li>ii. Annealing is a process where the forward primer and the reverse primer anneal to separate DNA template strands. </li></ul><ul><li>iii. Extension came with the enzyme called Taq polymerase which binds up all the nucleotides of the separated strand with its opposite partner. </li></ul><ul><li>One cycle was counted when all the three steps occurs. </li></ul>
  20. 44. PCR cont. <ul><li>Methodology </li></ul><ul><li>PCR screening was done by adding up a mixture of reagents necessary as mentioned: </li></ul><ul><ul><li>10x TE buffer </li></ul></ul><ul><li>MgCl 2 (25 mmol/l) </li></ul><ul><li>DNTPs (20 mmol/l) </li></ul><ul><li>Forward primer </li></ul><ul><li>Reverse primer </li></ul><ul><li>Taq polymerase </li></ul><ul><li>Template DNA </li></ul><ul><li>Sterile deionised distilled water </li></ul><ul><li>Mixture is then pipetted into an appendorf tube and mix gently. After mixing, add mineral oil and put inside the thermal cycler for 30 cycles. After amplification by PCR, the products are separated by agarose gel electrophoresis and are directly visualized after staining with ethidium bromide. </li></ul>
  21. 45. Step 9: TOPO TA Cloning <ul><li>Principle </li></ul><ul><li>For ligation, we use the TOPO TA cloning method that combines the advantages of TA cloning with the ligation activity of topoisomerase I. This allows direct ligation of our PCR products in just 5 minutes. TA Cloning is a method that takes advantage of the terminal transferase activity of some DNA polymerase such as Taq polymerase. This enzyme adds a single, 3'-A overhang to each end of the PCR product. This makes it possible to clone this PCR product directly into a linearized cloning vector with single, 3'-T overhangs. DNA polymerases with proofreading activity, such as Pfu polymerase, cannot be used because they provide blunt-ended PCR products. For this purpose, we use pCR4-TOPO as our cloning vector, which is digested in two sequential reactions with Eco R1 gives a linearized vector with 3'-T overhangs and a low background of non-recombinants. </li></ul><ul><li>TOPO TA cloning kits are available from Invitrogen . </li></ul><ul><li>TA cloning kits are available from different manufacturers. </li></ul>
  22. 46. TOPO TA Cloning cont. <ul><li>The ligation process is shown by the flow chart below: </li></ul>
  23. 47. TRANSFORMATION INTO AN E.COLI. <ul><li>Uptake of DNA into ‘competent’ bacterial cell. </li></ul><ul><li>E.coli takes up only limited amount of DNA under normal conditions. </li></ul><ul><li>Bacteria have to undergo some form of physical and chemical treatment-enhance ability to take up DNA. Cells that undergo this treatment are called competent. </li></ul><ul><li>Competence is the ability to take up DNA from external environment </li></ul>
  24. 48. <ul><li>Ligation mix added into competent cells and kept on ice for 20 minutes </li></ul><ul><li>The tube is kept on 42 0 C water bath for 30 seconds (heat shock treatment) and kept in ice for 2 min. </li></ul><ul><li>Add 350µl LB broth and incubate on 37 0 C and plate 200µl on LB Ampicilin agar plates using L glass rod and incubate overnight at 37 0 C </li></ul>PROCEDURES
  25. 49. VERIFICATION OF SUCCESSFUL TRANSFORMATION <ul><li>Once transformation has been performed, it is then necessary to distinguish cells that have been transformed, or were transformed with contaminating uncut vector. </li></ul><ul><li>The vector system that is being employed here manages this using antibiotic resistance Amp R encoded on the vector plasmid. Only those cells that have received plasmid will be capable of growing in the presence of this antibiotic. </li></ul><ul><li>Thus, we can identify the vector, pCR4-TOPO that has been successfully inserted with gene erythropoietin. </li></ul><ul><li>Some other vectors employ a colour selection system allowing an inserted piece of DNA </li></ul>
  26. 50. Con’t <ul><li>LacZ gene is inactivated by insertion of DNA into this site. When inactivated by inserted DNA, this gene is no longer producing  -galactosidase, no blue product released, colonies resulting from cells that have been transformed with vector only and will grow on the plates containing an ampicilin resistance gene that are introduced into E.coli cells by transformation. </li></ul>
  27. 51. Con’t
  28. 52. Step 11: Screening of Recombinant Clone through PCR Method
  29. 56. PROTEIN PURIFICATION <ul><li>Nickel metal affinity chromatography method </li></ul><ul><li>* separate the proteins based on the affinity between the neighboring histidines of the His Tag sequence and an immobilized metal ion (usually Ni +2 ). </li></ul>
  30. 57. PROTEIN PURIFICATION nickel metal affinity chromatography method
  31. 58. PROTEIN PURIFICATION <ul><li>recombinant protein is attached with 6 Histidine tag </li></ul><ul><li>passing through a nickel affinity column </li></ul><ul><li>target protein will bind with the nickel that is attached to the resin </li></ul><ul><li>unbounded protein or protein without histidine will be washed away through the column with the buffer </li></ul><ul><li>pH of the buffer is lowered by adding elution buffer (pH5.6) to reduce the binding affinity of the protein to the resin </li></ul>
  32. 59. <ul><li>target protein is flowed down through the column </li></ul><ul><li>His tag protein is then treated with the specific protease (in this case enterokinase) to cleave off the His Tag </li></ul><ul><li>The His Tag is detached from protein. </li></ul><ul><li>the recombinant protein is freed of the His Tag peptide by running it over the metal-column. The His Tag binds to the nickel column and the target protein flows down. </li></ul><ul><li>Pure protein is obtained and is anlyzed next with SDS- page </li></ul>PROTEIN PURIFICATION
  33. 60. SDS PAGE
  34. 61. SDS PAGE <ul><li>SDS page is now the most widely used technique to analyse protein mixtures.The anionic detergent SDS is a very effective solubilizing agent for a wide range of protein </li></ul><ul><li>Proteins are thus separated on the basis of size by the sieving effect of the polyacrylamide gel matrix </li></ul><ul><li>Protein are generally denatured and solubilised.The SDS-protein complex forms a rod (linear) proportional to its molecular weight. </li></ul>
  35. 62. SDS PAGE <ul><li>Properties of the polyacrylamide gel include formation of the cross linking agent (N,N-methylene bisacrylamide (BIS) to form a 3D lattice. Polymerization is initiated by TEMED (N,N,N1-tetramethylethylenediamine) and catalysed by ammonium persulfate </li></ul><ul><li>Discontinuous gel-consist of stacking (upper gel) and resolving gel(lower gel). </li></ul>
  36. 63. SDS PAGE-cont; <ul><li>The stacking gel will concentrate large sample volumes resulting in a better band resolution. </li></ul><ul><li>Molecules are then completely separated using the resolving gel. Laemli buffer is used. </li></ul>
  37. 65. SDS PAGE <ul><li>An estimate of the molecular weight of the unknown can be done by comparing against the electrophoretic mobility of the known molecular weight markers.An Rf graph can be drawn using the known molecular weights of the unknown are read off the graph. </li></ul><ul><li>Proteins separated by PAGE can be visualised using staining method with Coomassie Brilliant blue R250. The gel are then destained to observe the protein bands. </li></ul>
  38. 66. COMMERCIAL VALUES <ul><li>Uses of Erythropoietin </li></ul><ul><li>treat anemia </li></ul><ul><li>protect against nerve damage </li></ul><ul><ul><li>therapy for stroke and spinal cord injury </li></ul></ul><ul><ul><li>prevents motor neuron apoptosis and neurological disability in spinal cord and ischemic injury </li></ul></ul><ul><ul><li>protect against brain injury </li></ul></ul>
  39. 67. References <ul><ul><ul><li> </li></ul></ul></ul><ul><ul><ul><li> </li></ul></ul></ul><ul><ul><ul><li> </li></ul></ul></ul><ul><ul><ul><li> </li></ul></ul></ul><ul><ul><ul><li> </li></ul></ul></ul><ul><ul><ul><li> </li></ul></ul></ul>