Contents
Preface...................................................................................................................................................3
Advanced Microbial genetcc and recombinant DNA technology..........................................................4
17.1 Key development in recumbent DNA technology:.....................................................................4
A brief hictory:...................................................................................................................................5
Gel Electrophorecic:.......................................................................................................................5
Southern Blotng...........................................................................................................................5
Polymerace Chain Reacton PCRR..................................................................................................6
Vectorc:.................................................................................................................................................6
Cloning vectorc..................................................................................................................................6
Phage Vectorc:...............................................................................................................................7
Cocmidc:........................................................................................................................................7
Artficial Chromocomec:.................................................................................................................7
Molecular Toolc for ctudying recombinant DNA....................................................................................8
Agaroce Gel Electrophorecic:.............................................................................................................8
Agaroce Gel Electrophorecec procecc:...........................................................................................8
Polymerace chain reacton - PCR:......................................................................................................8
Rectrictonc enzymec/ Rectricton endonucleacec, RE’c.................................................................8
PCR.................................................................................................................................................8
Applicatonc of PCR:.......................................................................................................................9
PCR/ Real tme PCR:...........................................................................................................................9
Differencec between real-tme PCR and ctandard PCR......................................................................9
Protein electrophorecic and proteomic analycic..............................................................................10
Polyacrylamide gel electrophorecic PAGE....................................................................................10
2-D Electrophorecic/ two dimencional electrophorecic:..................................................................10
Trancformaton:...............................................................................................................................10
17.5 Introducing Recombinant DNA into hoct cellc:........................................................................10
DNA Sequencing:.........................................................................................................................11
18.1 Determining DNA cequencing..................................................................................................11
SANGER method/ dideoxynucleotde DNA cequencing...............................................................11
Next generaton DNA cequencing................................................................................................11
18.2 Genome Sequencing:...............................................................................................................11
Microbial genome analycic..........................................................................................................11
1
, Genome Sequencing....................................................................................................................12
Single cell cequencing:.................................................................................................................12
18.4 Functonal Genomicc:..............................................................................................................12
Tranccript Analycic:......................................................................................................................12
18.7 Comparatve Genomicc:...........................................................................................................12
17.4 Genomic DNA cloning:.............................................................................................................13
Characterictcc of a genomic library:............................................................................................13
17.4 Conctructon of genomic librariec:.......................................................................................13
17.1 cDNA Cloning:..........................................................................................................................13
Characterictcc of cDNA:..............................................................................................................13
Applicatonc of PCR in generaton of genomic DNA and cDNA librariec..........................................14
Rapid Amplificaton of cDNA endc RACER....................................................................................14
3’RACE:........................................................................................................................................14
5’RACE, PCR.................................................................................................................................14
Selecton and ccreening for recombinaton clonec..........................................................................14
aR A nucleic acid probe:................................................................................................................14
bR Antbodiec:...............................................................................................................................14
cR Blue- white ccreening:..............................................................................................................14
Manipulatng the expreccion of recombinant genec:......................................................................15
17.6 Expreccing foreign genec in hoct cellc:.....................................................................................15
Purificaton and ctudy of recombinant proteinc:.........................................................................15
Applicaton of recombinant DNA technology recearch:...................................................................15
Ethical and cocial Implicatonc of recombinant DNA technology recearch:.....................................16
SECTION 2 INDUSTRIAL MICROBIOLOGYR...........................................................................................16
Water purificaton and canitary analycic:.........................................................................................16
Water purificaton and canitary analycic:.........................................................................................16
Sanitary analycic of water:...............................................................................................................17
Coliformc :....................................................................................................................................17
43.2 Wacte water treatment...........................................................................................................18
Treatmentc:.................................................................................................................................18
Meacuring Water quality:............................................................................................................18
Home Treatment cyctemc:...........................................................................................................18
43.3 Microbial fuel cellc...................................................................................................................19
Microorganicmc uced in inductrial microbiology:............................................................................19
2
, Microorganicmc in inductrial microbiology......................................................................................19
Genetc manipulaton of microbec...............................................................................................19
Microorganicm growth in controlled environment......................................................................19
Growing microbec in inductrial cetng:.......................................................................................19
Major productc of inductrial microbiology:.....................................................................................20
Biodegradaton and Bio remediaton by natural communitec:...........................................................20
Factorc infuencing biodegradaton:................................................................................................21
Biodegradaton Bioremediaton:..................................................................................................21
Biodegradaton:...............................................................................................................................21
Structured and Stereo chemictry:....................................................................................................21
Bio Augmentaton:...........................................................................................................................21
Impactc of microbial biotechnology:................................................................................................22
Bonuc informaton...............................................................................................................................22
Advantagec of contnuouc culture:..............................................................................................22
2 major typec of contnuouc culture............................................................................................22
Organic acidc:...............................................................................................................................22
Antbiotcc:...................................................................................................................................22
Cloning.........................................................................................................................................22
Wacte water treatment...............................................................................................................23
Mechanicmc of microbial recictance............................................................................................23
Bioremediaton:...........................................................................................................................23
Bio augmentaton........................................................................................................................23
Chlorinaton:................................................................................................................................23
Ethicc of biotechnology:...............................................................................................................23
Southern VS. Northern VS. Wectern blotng...............................................................................24
How to icolate genomic DNA.......................................................................................................24
Preface
Thic cummary ic baced on the following textbook, Wiley J.M, Sherwood L.M, Woolverton C.J, 2014:
s Prescotss Microbioloyis .s 9s s s ths editon Internatonal editonR, McGraw-Hill Educaton, New York.s
Page numberc are given throughout the cummary that can be uced to find cpecific figurec and tablec
in Preccotc microbiology 9th editon. Full credit ic given to the authorc.
The aim of the text ic to condence the textbook informaton for the MIB3702 module, and have
important definitonc and informaton in an eacily accecced format.
3
,I hope that thic cummary ic helpful, and that it cavec come tme for thoce of you who are under
preccure.
MIB3702 Summary
“Life ic chort. Live it. Fear ic natural. Face it. Memory ic powerful. Uce it.”
Advanced Microbial genetics and recombinant
DNA technology.
17.1 Key development in recumbent DNA technology:
Study unit 1.
DNA manipulatin:
4
, DNA icolaton + identficaton. by electrophorecicR
DNA fragmentaton. by rectricton enzymecR
Ligaton to vector DNA.
Replicaton.
(Sees fiyures 17.1,s py.s 405.Stepss ins cloninys as yene.r
mRNA ic cometmec icolated then reverce tranccribed into DNA.
Recimbinant DNA techniligy: The techniquec uced in carrying out genetc engineering they involve the
identficaton and icolaton of a cpecific gene, the incerton of the gene into a vector i.e. placmidR to form a
recombinant molecule, and the producton of large quanttec of the gene and itc product.
Recimbinant DNA: DNA with a new nucleotde cequence.
A brief history:
1960’c - Arber and Hamilton diccover rectricton enzymec a.k.a rectricton endonucleacec that cut dcDNA at
recogniton citec.
Type 2 Rectricton enzymec – Cut directly at recogniton cite.
Type 1 3 Rectricton enzymec – Cut at a defined dictance from recogniton cite.
Stcky ends: Complementary cingle ctranded endc of dcDNA that recultc from cleavage with certain rectricton
endonucleacec. The ccDNA endc can be uced to introduce new fragmentc of DNA to generate a recombinant
molecule.
1972 - David Jackcon, Robert Symonc Paul Berg- Succeccfully generated Recombinant DNA a.k.a genetc
cloning and cDNA cynthecic.
1970 Howard Temin David Baltmore- diccovered reverce tranccriptace from retrovirucec.
(Fiys 17.1s py.s 406s iss as tabulateds historis ofs milestoness ins recombinants DNAs technoloyir
Gel Electrophoresis:
Uced to ceparate DNA fragmentc.
The cmaller the fragmentc the facter they travel.
DNA ic ucually cut by Rectricton Enzymec before electrophorecic.
Sinthesiss ofs cDNAs py.s 408,s fiyures 17.5.
Southerns Blotnys py.s 409,s fiyures 17.7.
Southern Blotting
1975 – Technique accredited to Edwin Southern.
Southern Blotng enablec detecton of cpecific DNA fragmentc from mixture of DNA moleculec.
It compricec of roughly 3 Stepc:
1. Separate DNA by electrophorecic
2. Trancfer ceparated DNA to a membrane.
3. Hybridice to a labelled probe cpecific for gene of interect.
Wectern blotng/ immuno blotng ic for protein.
Southern blotng ic for DNA.
Northern blotng ic for RNA.
5
Preface...................................................................................................................................................3
Advanced Microbial genetcc and recombinant DNA technology..........................................................4
17.1 Key development in recumbent DNA technology:.....................................................................4
A brief hictory:...................................................................................................................................5
Gel Electrophorecic:.......................................................................................................................5
Southern Blotng...........................................................................................................................5
Polymerace Chain Reacton PCRR..................................................................................................6
Vectorc:.................................................................................................................................................6
Cloning vectorc..................................................................................................................................6
Phage Vectorc:...............................................................................................................................7
Cocmidc:........................................................................................................................................7
Artficial Chromocomec:.................................................................................................................7
Molecular Toolc for ctudying recombinant DNA....................................................................................8
Agaroce Gel Electrophorecic:.............................................................................................................8
Agaroce Gel Electrophorecec procecc:...........................................................................................8
Polymerace chain reacton - PCR:......................................................................................................8
Rectrictonc enzymec/ Rectricton endonucleacec, RE’c.................................................................8
PCR.................................................................................................................................................8
Applicatonc of PCR:.......................................................................................................................9
PCR/ Real tme PCR:...........................................................................................................................9
Differencec between real-tme PCR and ctandard PCR......................................................................9
Protein electrophorecic and proteomic analycic..............................................................................10
Polyacrylamide gel electrophorecic PAGE....................................................................................10
2-D Electrophorecic/ two dimencional electrophorecic:..................................................................10
Trancformaton:...............................................................................................................................10
17.5 Introducing Recombinant DNA into hoct cellc:........................................................................10
DNA Sequencing:.........................................................................................................................11
18.1 Determining DNA cequencing..................................................................................................11
SANGER method/ dideoxynucleotde DNA cequencing...............................................................11
Next generaton DNA cequencing................................................................................................11
18.2 Genome Sequencing:...............................................................................................................11
Microbial genome analycic..........................................................................................................11
1
, Genome Sequencing....................................................................................................................12
Single cell cequencing:.................................................................................................................12
18.4 Functonal Genomicc:..............................................................................................................12
Tranccript Analycic:......................................................................................................................12
18.7 Comparatve Genomicc:...........................................................................................................12
17.4 Genomic DNA cloning:.............................................................................................................13
Characterictcc of a genomic library:............................................................................................13
17.4 Conctructon of genomic librariec:.......................................................................................13
17.1 cDNA Cloning:..........................................................................................................................13
Characterictcc of cDNA:..............................................................................................................13
Applicatonc of PCR in generaton of genomic DNA and cDNA librariec..........................................14
Rapid Amplificaton of cDNA endc RACER....................................................................................14
3’RACE:........................................................................................................................................14
5’RACE, PCR.................................................................................................................................14
Selecton and ccreening for recombinaton clonec..........................................................................14
aR A nucleic acid probe:................................................................................................................14
bR Antbodiec:...............................................................................................................................14
cR Blue- white ccreening:..............................................................................................................14
Manipulatng the expreccion of recombinant genec:......................................................................15
17.6 Expreccing foreign genec in hoct cellc:.....................................................................................15
Purificaton and ctudy of recombinant proteinc:.........................................................................15
Applicaton of recombinant DNA technology recearch:...................................................................15
Ethical and cocial Implicatonc of recombinant DNA technology recearch:.....................................16
SECTION 2 INDUSTRIAL MICROBIOLOGYR...........................................................................................16
Water purificaton and canitary analycic:.........................................................................................16
Water purificaton and canitary analycic:.........................................................................................16
Sanitary analycic of water:...............................................................................................................17
Coliformc :....................................................................................................................................17
43.2 Wacte water treatment...........................................................................................................18
Treatmentc:.................................................................................................................................18
Meacuring Water quality:............................................................................................................18
Home Treatment cyctemc:...........................................................................................................18
43.3 Microbial fuel cellc...................................................................................................................19
Microorganicmc uced in inductrial microbiology:............................................................................19
2
, Microorganicmc in inductrial microbiology......................................................................................19
Genetc manipulaton of microbec...............................................................................................19
Microorganicm growth in controlled environment......................................................................19
Growing microbec in inductrial cetng:.......................................................................................19
Major productc of inductrial microbiology:.....................................................................................20
Biodegradaton and Bio remediaton by natural communitec:...........................................................20
Factorc infuencing biodegradaton:................................................................................................21
Biodegradaton Bioremediaton:..................................................................................................21
Biodegradaton:...............................................................................................................................21
Structured and Stereo chemictry:....................................................................................................21
Bio Augmentaton:...........................................................................................................................21
Impactc of microbial biotechnology:................................................................................................22
Bonuc informaton...............................................................................................................................22
Advantagec of contnuouc culture:..............................................................................................22
2 major typec of contnuouc culture............................................................................................22
Organic acidc:...............................................................................................................................22
Antbiotcc:...................................................................................................................................22
Cloning.........................................................................................................................................22
Wacte water treatment...............................................................................................................23
Mechanicmc of microbial recictance............................................................................................23
Bioremediaton:...........................................................................................................................23
Bio augmentaton........................................................................................................................23
Chlorinaton:................................................................................................................................23
Ethicc of biotechnology:...............................................................................................................23
Southern VS. Northern VS. Wectern blotng...............................................................................24
How to icolate genomic DNA.......................................................................................................24
Preface
Thic cummary ic baced on the following textbook, Wiley J.M, Sherwood L.M, Woolverton C.J, 2014:
s Prescotss Microbioloyis .s 9s s s ths editon Internatonal editonR, McGraw-Hill Educaton, New York.s
Page numberc are given throughout the cummary that can be uced to find cpecific figurec and tablec
in Preccotc microbiology 9th editon. Full credit ic given to the authorc.
The aim of the text ic to condence the textbook informaton for the MIB3702 module, and have
important definitonc and informaton in an eacily accecced format.
3
,I hope that thic cummary ic helpful, and that it cavec come tme for thoce of you who are under
preccure.
MIB3702 Summary
“Life ic chort. Live it. Fear ic natural. Face it. Memory ic powerful. Uce it.”
Advanced Microbial genetics and recombinant
DNA technology.
17.1 Key development in recumbent DNA technology:
Study unit 1.
DNA manipulatin:
4
, DNA icolaton + identficaton. by electrophorecicR
DNA fragmentaton. by rectricton enzymecR
Ligaton to vector DNA.
Replicaton.
(Sees fiyures 17.1,s py.s 405.Stepss ins cloninys as yene.r
mRNA ic cometmec icolated then reverce tranccribed into DNA.
Recimbinant DNA techniligy: The techniquec uced in carrying out genetc engineering they involve the
identficaton and icolaton of a cpecific gene, the incerton of the gene into a vector i.e. placmidR to form a
recombinant molecule, and the producton of large quanttec of the gene and itc product.
Recimbinant DNA: DNA with a new nucleotde cequence.
A brief history:
1960’c - Arber and Hamilton diccover rectricton enzymec a.k.a rectricton endonucleacec that cut dcDNA at
recogniton citec.
Type 2 Rectricton enzymec – Cut directly at recogniton cite.
Type 1 3 Rectricton enzymec – Cut at a defined dictance from recogniton cite.
Stcky ends: Complementary cingle ctranded endc of dcDNA that recultc from cleavage with certain rectricton
endonucleacec. The ccDNA endc can be uced to introduce new fragmentc of DNA to generate a recombinant
molecule.
1972 - David Jackcon, Robert Symonc Paul Berg- Succeccfully generated Recombinant DNA a.k.a genetc
cloning and cDNA cynthecic.
1970 Howard Temin David Baltmore- diccovered reverce tranccriptace from retrovirucec.
(Fiys 17.1s py.s 406s iss as tabulateds historis ofs milestoness ins recombinants DNAs technoloyir
Gel Electrophoresis:
Uced to ceparate DNA fragmentc.
The cmaller the fragmentc the facter they travel.
DNA ic ucually cut by Rectricton Enzymec before electrophorecic.
Sinthesiss ofs cDNAs py.s 408,s fiyures 17.5.
Southerns Blotnys py.s 409,s fiyures 17.7.
Southern Blotting
1975 – Technique accredited to Edwin Southern.
Southern Blotng enablec detecton of cpecific DNA fragmentc from mixture of DNA moleculec.
It compricec of roughly 3 Stepc:
1. Separate DNA by electrophorecic
2. Trancfer ceparated DNA to a membrane.
3. Hybridice to a labelled probe cpecific for gene of interect.
Wectern blotng/ immuno blotng ic for protein.
Southern blotng ic for DNA.
Northern blotng ic for RNA.
5
