Molecular cloning

Molecular re-createAbstr doingMolecular cl whizz is a method to produce quantities of a specific deoxyribonucleic acid segment. It contains an abundance of techniques including DNA transfer, DNA recombination, DNA sequencing and so on. Though this practical, labour maps were draw for plasmid DNA pMA and pMB by doing single and double digest, a pMB segment digested with PstI was innovateed to plasmid pUC19 and then transferred to host cells to have proliferation and expression, and the eon of PstI digested pMB break up was analysed.1. Introduction Recombinant DNA molecules ar molecules containing DNA sequences derived from more than one source. In molecular cloning, by utilize recombinant DNA, a specific combination of factors put forward be put into a carrier, and then basin be proliferated and expressed in a telephone receiver cell. In medicine, by making use of molecular cloning, scientists have successfully constructed engineering strains of insulin, egress hormone of human, cattle and chicken, human interferon, erythropoietin, antigen of hepatitis B computer virus and antigen of foot-and-mouth disease virus, and conducted a large-scale production by fermentation industry. In gene therapy, there is a possibility of reversing crabmeat cells to normal cells by dint of genetic engineering, for example, mouse tumor cells caused by SV40 virus can turnround to normal cells at high temperature. Many chemical reagents such as acrylic acid, ethylene glycol, methanol, ethylene oxide and salicylic acid can possibly be produced by making use of molecular cloning. In environmental protection, mass transfer genes of one microorganism into another through genetic manipulation to create new strains that are more capable of degrading bad substances, in order to break down toxic substances in industrial waste.1,2Blue-White selection is a method for screening recombinant DNA. Vectors containing a -galactosidase gene (lacZ) can have a complementation (-comp lementation) with E.coli strain to form a serviceable -galactosidase enzyme. Neither vectors, nor host cells have the enzyme activity. The lacZ gene has an internal multiple cloning sites (MCS) which can be dilute by different barricade enzymes. Therefore, when a gene sever is inserted in the vector, the lacZ gene will be break up and cannot form active -galactosidase enzyme. X-gal can be metabolized by -galactosidase to gain a full-bodied product. Therefore, in the presence of X-gal, DNA with no insert gene can display a blue colour, while recombinant DNA, which have no enzyme function, display a white colour.3The aim of the practical is to draw confinement maps of simple plasmids for recombinant DNA, do basic molecular cloning and sequence a DNA fragment.2. ResultsTable 1 Antibiotic resistances of 5 E. coli strainsLB/ampicillinLB/TetracyclineLB/KanamycinDH5aNo branchNo growthNo growthpUC19GrownNo growthNo growthpMAGrownGrownNo growthpMBNo growthGrownGrownXL1-BlueNo growthG rownNo growthDH5a E. coli strain DH5a pUC19 E. coli strain DH5a containing plasmid pUC19 pMA E. coli strain DH5a containing plasmid pMA pMB E. coli strain DH5a containing plasmid pMB XL1-Blue E. coli strain XL1-Blue.NO.DNAEnzyme1pMABam HI2pMAXhoI3pMAPstI4pMAEcoRI5pMBBam HI6pMBXhoI7pMBPstI8pMBEcoRI9Lambda soft touch10X174 markerNO.DNAEnzymes1pMAEcoRI, Bam HI2pMAEcoRI, PstI3pMAEcoRI, XhoI4pMABam HI, PstI5pMABam HI, XhoI6pMAPstI, XhoI7pMBEcoRI, Bam HI8pMBEcoRI, PstI9pMBEcoRI, XhoI10pMBBam HI, PstI11pMBBam HI, XhoI12pMBPstI, XhoI13Lambda marker14X174 marker1Lambda marker2Blue resolution digested with PstI3-7White colonies digested with PstI8X174 marker gagtantagttcgccngttaatagtttgcgcaacgttgttgccattgctgcaggggggggggggaaagccacgttgtgtctcaaaatctctgatgttacattgcacaagataaaaatatatcatcatgaacaataaaactgtctgcttacataaacagtaatacaaggggtgttatgagccatattcaacgggaaacgtcttgctcgaggccgcgattaaattccaacatggatgctgatttatatgggtataaatgggctcgcgataatgtcgggcaatcaggtgcgacaatctatcgattgtatgggaagcccgatgcgccagagttgtttctgaa acatggcaaaggtagcgttgccaatgatgttacagatgagatggtcagactaaactggctgacggaatttatgcctcttccgaccatcaagcattttatccgtactcctgatgatgcatggttactcaccactgcgatccccgggaaaacagcattccaggtattagaagaatatcctgattcaggtgaaaatattgttgatgcgctggcagtgttcctgcgccggttgcattcgattcctgtttgtaattgtccttttaacagcgatcgcgtatttcgtctcgctcaggcgcaatcacgaatgaataacggtttggttgatgcgagtgattttgatgacgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataagcttttgccattctcaccggattcagtcgtcactcatggtgatttctcacttgatgaggttatttttgacgaggggaaattaataggttgtattgatgttggacgagtcggaatcgcagaccgataccaggatcttgctttttcaaaaatatggtattgataatcctgatatgaataaattgcagtttcatttgatgctcgatgagtttttttaatgagaattggttaattggttgtaacactggcagagcattacgctgacttgacgggacggcggctttgttgaataaatcgaacttttgctgagttgaaggatcagatcacgcatcttcccgacaacgcagaccgttccgtggcaaagcaaaagttcaaaatcaccaactggtccacctacaacaaagctctcatcaaccgtggctccctcactttctggctggatgatggggcgattcaggcctcaacgactgagtatggaccttcttcacgaggcagacctcagcgccccccccccccctgcaggcaEnzymeNo. of cutsPosition of sites (bp)Recognition sequencePstI252, 1243ctgca/gXhoI1204c /tcgagE X plosive speech sound F A X immobilize _ F A Q R C C H C C R G G G E S H V V S Q N L Stop C Y I A Q D K N I S S Stop T I K L S A Y I N S N T R G V M S H I Q R E T S C S R P R L N S N M D A D L Y G Y K W A R D N V G Q S G A T I Y R L Y G K P D A P E L F L K H G K G S V A N D V T D E M V R L N W L T E F M P L P T I K H F I R T P D D A W L L T T A I P G K T A F Q V L E E Y P D S G E N I V D A L A V F L R R L H S I P V C N C P F N S D R V F R L A Q A Q S R M N N G L V D A S D F D D E R N G W P V E Q V W K E M H K L L P F S P D S V V T H G D F S L D E V I F D E G K L I G C I D V G R V G I A D R Y Q D L A F S K I W Y Stop _ S Stop Y E Stop I A V S F D A R Stop V F L M R I G Stop L V V T L A E H Y A D L T G R R L C Stop I N R T F A E L K D Q I T H L P D N A D R S V A K Q K F K I T N W S T Y N K A L I N R G S L T F W L D D G A I Q A S T T E Y G P S S R G R P Q R P P P P C RAminoglycoside 3-phosphotransferase, putative3. Discussion3.1 Antibiotics resistances Seen from remand 1, D H5a has no resistance to any of the cardinal bacteria, pUC19 is yucky to ampicillin, pMA is resistant to ampicillin and tetracycline, pMB is resistant to tetracycline and Kanamycin, and XL1-Blue is resistant to tetracycline. Plasmid pUC19, pMA and pMB, which were used in the cloning procedure, had different antibiotic drug resistances, while the bacterial host, DH5a, have no antibiotic resistance. Therefore, cells containing recombinant DNA could be selected by growing host cells in presence of antibiotic. Even when different plasmids are contained in the host cells, this method can be used. For example, tetracycline can be used to select cells containing only(prenominal) pMA from a mixture of cells containing pMA and pUC19.3.2 Restriction maps and relationship of pMA and pMB From the single digest (1), pMA could be cut by Bam HI, PstI and EcoRI, and each enzyme could cut pMA once. However, pMA could not be cut by XhoI. pMB could be cut by Bam HI, XhoI and EcoRI once, and cut by PstI twice. Therefore, pMA has three rampart enzyme sites, while pMB has five. From the double digest (2), the results were consistent with single digest, and the continuance of each fragment could be obtained. Restriction maps (3) were drawn storyd on the single and double digests. From the lying-in maps, the fragments in pMA and pMB, cutting by Bam HI and EcoRI, have the same base pairs (430bp). The fragment cutting by EcoRI and PstI in pMA has the same base pair (720bp) with one of the fragments cutting by EcoRI and PstI in pMB. The fragment cutting by Bam HI and PstI in pMA has the same base pair (1150bp) with one of the fragments cutting by EcoRI and PstI in pMB. The continuing fragment in pMB cutting by PstI was round about 3780bp, which was very constrictive to the length of pMA (3800bp).As all the lengths of fragments were roughly obtained and were not accurate. Therefore, we can espouse that pMA is a part of pMB. pMB can be cut by PstI. If the longer fragment is re- circled, it will have the same base pairs and restriction enzyme sites (PstI, EcoRI and Bam HI) with pMA. The XhoI restriction site on pMB is between the two restriction sites of PstI, therefore, the longer fragment cannot be cut by XhoI, which is consistent with pMA. Seen from the antibody resistances, pMA is resistant to ampicillin and tetracycline, pMB is resistant to tetracycline and Kanamycin. This might because the tetracycline resistant gene is in pMA, which is a part of pMB. And kanamycin resistant gene is in the PstI fragment of pMB, which pMA does not have. For the ampicillin resistant gene, it might be located around the PstI restriction site in pMA, which will be insertion inactive when insert the PstI fragment to pMA to make it become pMB, therefore, pMB does not have ampicillin resistance.This possibleness can be proved by sequencing pMA and pMB fragment cutting by PstI, which was not included in this experiment.3.3 Sub cloning recombinant clonesIn 4, the blue colony had only one flock, which meant that there was only one PstI restriction site in the plasmid. This was consistent with pUC19 that did not have an insert fragment. quad of the white colonies had two bands each, including one band located around 1200bp. These were the recombinant DNA, with the pMB fragment digested with PstI. One white colony (No. 7) did not have a band located around 1200bp, but a fragment shorter than that. This was also a recombinant DNA, with other fragment rather than PstI fragment. This might be caused by about impurities through the procedure.3.4 Sequence analysisThe sequence of the PstI fragment in pMB was obtained by everywherelapping two fragments (forward and reverse). Seen from 5, there are two PstI restriction sites (ctgca/g) and one XhoI restriction site (c/tcgag), and the XhoI restriction site is between the two PstI restriction sites. Therefore, if the fragment is digested with PstI and XhoI, two fragments (152bp, 1039bp). This is roughly consistent with the restriction map of pMB which was not accurate.The aminic acid sequence shown in 6 is one of the 6 possible sequences (53 Frame 1), methionine, which is a start of protein sequence, and stop codons are over striking. One potentially matching sequence of protein encoded in the PstI fragment of pMB shown in 7, aminoglycoside 3-phosphotransferase, begins with the first methionine in the fragment and have a length of 253 amino acids.4. Conclusion This practical provide us a better collar of how to make a recombinant DNA and molecular cloning technique. These experiences can act as fundament of further researches such as researches in cancer cells.References1 Williams Wu, Michael J. Welsh, et al. (2003) cistron Biotechnology (2nd edition).2 Gerald Karp. (2002) Cell and Molecular Biology (3rd edition).3 Benjamin Lewin. (2004) Gene (International edition).