Microbial genetics and genetic engineering

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  • slide 10
    difference of blunt end and a sticky end.

    blunt ends make create an unstable bond with the insert gene and vector. blunt ends may exhibit reformation of the bond, particularly, the vector may close the cut, calling it re-ligation. It also has the risk of reversing the orientation of the insert gene. (JUST LOOK AT THE FIGURE CAREFULLY)

    sticky ends forms an overhang.. which you can see clearly and visibly in the figure. overhangs can be more efficient against re-ligation of vector DNA but sometimes there are additional step in order to prevent this we call it dephosphorylation. ^^
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  • in slide #8
    Properties of vector:
    point 1 ~ it must be able to replicate meaning the DNA vector must be recognized by the host cell and as well as it is stable. checking for mutations and stability is also important. there is an important region in each plasmid that can be recognized by the host cell, it is called the replication origin. So there must be a replication origin for an E. coli plasmid DNA which will be recognized by the E. coli host cell for it to be replicated.

    point 2 ~ this is what we call transformation, the transfer or introduction of recombinant DNA (vector DNA + insert gene) there different ways to achieve transformation, the most common is heat shock method. other methods are electroporation or by chemical means such as CaCl2.

    point 3 ~ yes, plating techniques with selection markers. for example antibiotic gene will mean resistance to antibiotic, another is insert gene that encodes for enzymes for carbohydrate utilization. anything that will exhibit visible effect on a tangible scale.
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  • in slide #7
    point #4 - DNA ligase is the enzyme used for joining the insert gene (gene of interest) and the vector DNA (i.e. plasmid DNA)
    the word 'insert' can be better be substituted by transformed or transfer. it will be confusing to used the word insert since it is used referring to gene of interest
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  • This events could be so complex when using natural and random mutagens and then selection. That is why scientists develop technology that can modify organism in directed and predetermined way
  • RE are enzymes that can cut
  • Restriction mapping??
  • Microbial genetics and genetic engineering

    1. 1. Part 3
    2. 2. Mutagen Gene transfer Genetic recombi nationSelection of mutants
    3. 3. Recombinant DNA technology, gene cloning
    4. 4. • Genetic engineering involves changing the genetic material in an organism to alter its traits or products• A recombinant DNA molecule contains DNA fragments spliced together from 2 or more organisms
    5. 5.  Stages of cloning experiment Elements:  Vectors  Restriction enzymes  Mechanism in joining the fragments  Selection or detection of successful cloning Gene library
    6. 6. 1. Joining DNA segment Vector 2. Providing milieu that allows propagationClones
    7. 7. 1. Remove bacterial DNA (plasmid).2. Cut the Bacterial DNA with “restriction enzymes”.3. Cut the DNA from another organism with “restriction enzymes”.4. Combine the cut pieces of DNA together with another enzyme and insert them into bacteria.5. Reproduce the recombinant bacteria.6. The foreign genes will be expressed in the bacteria.
    8. 8. 1. It must be able to replicate2. There must be some way to introduce vector DNA into the cell3. There must be some way of detecting its presence, preferably by plating techniquesMost common vectors are: Plasmid, phage λ and viruses
    9. 9.  Discovered in an experiment where bacteriophage lost its plaque formation in E.coli Enzymes that recognize a specific base sequence in a DNA molecule It makes two cuts, one in each strand, generating 3’-OH and 5’-P termini Types of termini produced:  Flush or blunt end  Cohesive or sticky end
    10. 10.  The number of cuts made in the DNA from specific organism is limited A particular restriction enzyme generates a unique family fragments from a DNA molecule
    11. 11. •BstEII pUC19 pUC19 •HindIII •HindIII •BstEII
    12. 12. Restriction maps show the relative location of a selection of restriction sites along linear or circular DNA. EcoRI HindIIIPstII BamHI HindIII BamHI PstII
    13. 13. Digests1: EcoRI2: HindIII3: EcoRI + HindIIIResultant Fragments: approximate sizes1: 3 kb, 5 kb2: 6 kb, 2 kb3: 2 kb, 1 kb, 5 kb,
    14. 14. BglII BglII BamHIBglII BamHI PstI +BamHI +PstI +PstI 5.2 4.2 3.6 3.5 3.3 2.6 1.7 1.7 1.4 1.4 1.2 1.2 1.2 1.0 0.9 1.0 0.7 0.5 0.3 0.3 0.3 BglII BamHI PstI BglII PstI0.3 0.7 2.6 0.9 0.5 1.2
    15. 15. A) 11, 6, 5B) 14,8C) 16,6A x B) 8, 6, 5, 3A x C) 11, 5, 5, 1B x C) 8, 8, 6
    16. 16.  Cohesive end Blunt end  Increasing the DNA concentration and addition of ligase  Addition of homopolymers  Using of linkers
    17. 17.  Antibiotic resistance marker  Insertional inactivation Electrophoresis hybridization
    18. 18.  collection of all of the vector molecules, each carrying a piece of the chromosomal DNA of the organism

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