1 objectives describe the steps in gene cloning by using plasmid as the vector
TRANSCRIPT
![Page 1: 1 Objectives describe the steps in gene cloning by using plasmid as the vector](https://reader035.vdocuments.site/reader035/viewer/2022062519/5697bfee1a28abf838cb98bf/html5/thumbnails/1.jpg)
1
Objectives
• describe the steps in gene cloning by using plasmid as the vector
![Page 2: 1 Objectives describe the steps in gene cloning by using plasmid as the vector](https://reader035.vdocuments.site/reader035/viewer/2022062519/5697bfee1a28abf838cb98bf/html5/thumbnails/2.jpg)
The steps for cloning:
1) Isolation
2) Splicing
3) Insertion
4) Transformation
5) Screening
![Page 3: 1 Objectives describe the steps in gene cloning by using plasmid as the vector](https://reader035.vdocuments.site/reader035/viewer/2022062519/5697bfee1a28abf838cb98bf/html5/thumbnails/3.jpg)
3
1. Isolation of DNA (gene)
Steps in Gene Cloning
2. Slicing with restriction enzymes
3. Insertion
4. Transformation & amplification (multiplication @ cloning)
5. Screening ; to identify bacterial cell containing recombinant plasmid Probing ; to identify bacterial cell containing recombinant plasmid with target gene
![Page 4: 1 Objectives describe the steps in gene cloning by using plasmid as the vector](https://reader035.vdocuments.site/reader035/viewer/2022062519/5697bfee1a28abf838cb98bf/html5/thumbnails/4.jpg)
4
isolation of plasmid DNA (from E. coli) and DNA containing gene of interest
- plasmid DNA as cloning vector
Isolation
♫ plasmid DNA carries ampR gene and lacZ gene
- ampR gene: antibiotic resistance gene - lacZ gene : encode for β-galactosidase
![Page 5: 1 Objectives describe the steps in gene cloning by using plasmid as the vector](https://reader035.vdocuments.site/reader035/viewer/2022062519/5697bfee1a28abf838cb98bf/html5/thumbnails/5.jpg)
5
Isolation
![Page 6: 1 Objectives describe the steps in gene cloning by using plasmid as the vector](https://reader035.vdocuments.site/reader035/viewer/2022062519/5697bfee1a28abf838cb98bf/html5/thumbnails/6.jpg)
6
cut open the plasmid DNA at restriction site which lies within lacZ gene
cut out the DNA into many DNA fragments; some of these fragments carry the gene of interest
- by using same restriction enzyme which cut at restriction sites containing same palindromic sequence to produce sticky ends
Slicing (cutting / cleave)
![Page 7: 1 Objectives describe the steps in gene cloning by using plasmid as the vector](https://reader035.vdocuments.site/reader035/viewer/2022062519/5697bfee1a28abf838cb98bf/html5/thumbnails/7.jpg)
Insertion
![Page 8: 1 Objectives describe the steps in gene cloning by using plasmid as the vector](https://reader035.vdocuments.site/reader035/viewer/2022062519/5697bfee1a28abf838cb98bf/html5/thumbnails/8.jpg)
after mixing, the DNA fragments and the cut plasmids form the complementary
pairs
they are then joined by DNA ligase
creating a mixture of rDNA molecules
![Page 9: 1 Objectives describe the steps in gene cloning by using plasmid as the vector](https://reader035.vdocuments.site/reader035/viewer/2022062519/5697bfee1a28abf838cb98bf/html5/thumbnails/9.jpg)
♦ note that the lacZ has becomenonfunctional
♦ cannot codefor β-galactosidase
![Page 10: 1 Objectives describe the steps in gene cloning by using plasmid as the vector](https://reader035.vdocuments.site/reader035/viewer/2022062519/5697bfee1a28abf838cb98bf/html5/thumbnails/10.jpg)
Transformation
bacteria containing recombinant plasmid CAN’T produce β-galactosidase
![Page 11: 1 Objectives describe the steps in gene cloning by using plasmid as the vector](https://reader035.vdocuments.site/reader035/viewer/2022062519/5697bfee1a28abf838cb98bf/html5/thumbnails/11.jpg)
the rDNA are reintroduced into the bacteria
bacterial cells are mixed with rDNAin the presence of cold calcium chloride
followed by heating; making the bacterial cell wall permeable to plasmids
![Page 12: 1 Objectives describe the steps in gene cloning by using plasmid as the vector](https://reader035.vdocuments.site/reader035/viewer/2022062519/5697bfee1a28abf838cb98bf/html5/thumbnails/12.jpg)
Transformation
![Page 13: 1 Objectives describe the steps in gene cloning by using plasmid as the vector](https://reader035.vdocuments.site/reader035/viewer/2022062519/5697bfee1a28abf838cb98bf/html5/thumbnails/13.jpg)
13
Transformation
produce diverse of bacteria :
• bacteria with recombinant plasmid (containing gene of interest) e.g. gene encode for insulin
• bacteria with recombinant plasmid (containing gene which encode for other protein) e.g. gene encode for human growth hormone
• bacteria with NON-recombinant plasmid
![Page 14: 1 Objectives describe the steps in gene cloning by using plasmid as the vector](https://reader035.vdocuments.site/reader035/viewer/2022062519/5697bfee1a28abf838cb98bf/html5/thumbnails/14.jpg)
Screening
i.e. detecting the bacteria containing rDNA
Procedure 1 Blue-white screening
![Page 15: 1 Objectives describe the steps in gene cloning by using plasmid as the vector](https://reader035.vdocuments.site/reader035/viewer/2022062519/5697bfee1a28abf838cb98bf/html5/thumbnails/15.jpg)
15
- to identify bacteria containing recombinant plasmid
Blue-white screening
bacteria is cultured on medium containing antibiotic (ampicillin) and sugar (X-gal)
Observation
ONLY bacteria with plasmid grow ; has ampicillin resistance (ampR) gene
![Page 16: 1 Objectives describe the steps in gene cloning by using plasmid as the vector](https://reader035.vdocuments.site/reader035/viewer/2022062519/5697bfee1a28abf838cb98bf/html5/thumbnails/16.jpg)
16
X-gal is used to identify colonies bacteria with recombinant plasmids
bacteria colonies WITHOUT recombinant plasmid will stain blue ; β-galactosidase is produced by functional lacZ gene (hydrolyze X-gal to yield blue product)
bacteria colonies WITH recombinant plasmid will stain white ; β-galactosidase is NOT produced because lacZ gene is NON-functional; X-gal is NOT hydrolyzed
![Page 17: 1 Objectives describe the steps in gene cloning by using plasmid as the vector](https://reader035.vdocuments.site/reader035/viewer/2022062519/5697bfee1a28abf838cb98bf/html5/thumbnails/17.jpg)
17
Probing (Nucleic acid hybridization)
- to identify bacteria with recombinant plasmid containing gene of interest (target gene)
Probing (Nucleic acid hybridization)
based on base pairing between gene of interest (e.g. gene encode for insulin) and other DNA molecule known as DNA probe (short & single-stranded; labeled with radioactive isotope or fluorescent tag)
![Page 18: 1 Objectives describe the steps in gene cloning by using plasmid as the vector](https://reader035.vdocuments.site/reader035/viewer/2022062519/5697bfee1a28abf838cb98bf/html5/thumbnails/18.jpg)
18
Probing (Nucleic acid hybridization)
a master plate is prepared
STEP 1
- contain colonies of bacteria with recombinant plasmid in the mean time, DNA probe is prepared
STEP 2
nitrocellulose filter paper is placed onto the master plate the filter paper is pressed against the bacterial colonies on the master plate ; bacterial colonies is transferred onto the filter paper
![Page 19: 1 Objectives describe the steps in gene cloning by using plasmid as the vector](https://reader035.vdocuments.site/reader035/viewer/2022062519/5697bfee1a28abf838cb98bf/html5/thumbnails/19.jpg)
19
the filter paper is treated with NaOH or heat - to denature (separate) the DNA; double helix DNA single stranded DNA
STEP 3
Probing (Nucleic acid hybridization)
STEP 4
DNA probe solution is added to the filter paper
- DNA probe will hybridize (base-pair with any complementary bases of single stranded DNA)
![Page 20: 1 Objectives describe the steps in gene cloning by using plasmid as the vector](https://reader035.vdocuments.site/reader035/viewer/2022062519/5697bfee1a28abf838cb98bf/html5/thumbnails/20.jpg)
20
the filter paper is washed to remove the excess, unbound DNA probe
STEP 5
then, the filter paper is laid on X-ray film – autoradiography technique
the film (autoradiograph) is developed
Probing (Nucleic acid hybridization)
STEP 6
the autoradiograph is compared with the master plate the colonies which contain gene of interest is identified
![Page 21: 1 Objectives describe the steps in gene cloning by using plasmid as the vector](https://reader035.vdocuments.site/reader035/viewer/2022062519/5697bfee1a28abf838cb98bf/html5/thumbnails/21.jpg)
21
![Page 22: 1 Objectives describe the steps in gene cloning by using plasmid as the vector](https://reader035.vdocuments.site/reader035/viewer/2022062519/5697bfee1a28abf838cb98bf/html5/thumbnails/22.jpg)
22
REMIND AGAIN
![Page 23: 1 Objectives describe the steps in gene cloning by using plasmid as the vector](https://reader035.vdocuments.site/reader035/viewer/2022062519/5697bfee1a28abf838cb98bf/html5/thumbnails/23.jpg)
23
Isolation
Slicing
![Page 24: 1 Objectives describe the steps in gene cloning by using plasmid as the vector](https://reader035.vdocuments.site/reader035/viewer/2022062519/5697bfee1a28abf838cb98bf/html5/thumbnails/24.jpg)
24
Insertion
![Page 25: 1 Objectives describe the steps in gene cloning by using plasmid as the vector](https://reader035.vdocuments.site/reader035/viewer/2022062519/5697bfee1a28abf838cb98bf/html5/thumbnails/25.jpg)
25
Transformation Screening
![Page 26: 1 Objectives describe the steps in gene cloning by using plasmid as the vector](https://reader035.vdocuments.site/reader035/viewer/2022062519/5697bfee1a28abf838cb98bf/html5/thumbnails/26.jpg)
26
Master plate
Probing
STEP 1
DNA probe
![Page 27: 1 Objectives describe the steps in gene cloning by using plasmid as the vector](https://reader035.vdocuments.site/reader035/viewer/2022062519/5697bfee1a28abf838cb98bf/html5/thumbnails/27.jpg)
27
Probing STEP 2
Contain NAOH or Heat
To denature DNA ,double to single
![Page 28: 1 Objectives describe the steps in gene cloning by using plasmid as the vector](https://reader035.vdocuments.site/reader035/viewer/2022062519/5697bfee1a28abf838cb98bf/html5/thumbnails/28.jpg)
28
Probing STEP 4
![Page 29: 1 Objectives describe the steps in gene cloning by using plasmid as the vector](https://reader035.vdocuments.site/reader035/viewer/2022062519/5697bfee1a28abf838cb98bf/html5/thumbnails/29.jpg)
29
Probing
STEP 5 STEP 6
![Page 30: 1 Objectives describe the steps in gene cloning by using plasmid as the vector](https://reader035.vdocuments.site/reader035/viewer/2022062519/5697bfee1a28abf838cb98bf/html5/thumbnails/30.jpg)
The steps for cloning:
1) Isolation
2) Splicing
3) Insertion
4) Transformation
5) Screening
![Page 31: 1 Objectives describe the steps in gene cloning by using plasmid as the vector](https://reader035.vdocuments.site/reader035/viewer/2022062519/5697bfee1a28abf838cb98bf/html5/thumbnails/31.jpg)
![Page 32: 1 Objectives describe the steps in gene cloning by using plasmid as the vector](https://reader035.vdocuments.site/reader035/viewer/2022062519/5697bfee1a28abf838cb98bf/html5/thumbnails/32.jpg)
![Page 33: 1 Objectives describe the steps in gene cloning by using plasmid as the vector](https://reader035.vdocuments.site/reader035/viewer/2022062519/5697bfee1a28abf838cb98bf/html5/thumbnails/33.jpg)
![Page 34: 1 Objectives describe the steps in gene cloning by using plasmid as the vector](https://reader035.vdocuments.site/reader035/viewer/2022062519/5697bfee1a28abf838cb98bf/html5/thumbnails/34.jpg)
![Page 35: 1 Objectives describe the steps in gene cloning by using plasmid as the vector](https://reader035.vdocuments.site/reader035/viewer/2022062519/5697bfee1a28abf838cb98bf/html5/thumbnails/35.jpg)
![Page 36: 1 Objectives describe the steps in gene cloning by using plasmid as the vector](https://reader035.vdocuments.site/reader035/viewer/2022062519/5697bfee1a28abf838cb98bf/html5/thumbnails/36.jpg)
![Page 37: 1 Objectives describe the steps in gene cloning by using plasmid as the vector](https://reader035.vdocuments.site/reader035/viewer/2022062519/5697bfee1a28abf838cb98bf/html5/thumbnails/37.jpg)
![Page 38: 1 Objectives describe the steps in gene cloning by using plasmid as the vector](https://reader035.vdocuments.site/reader035/viewer/2022062519/5697bfee1a28abf838cb98bf/html5/thumbnails/38.jpg)
![Page 39: 1 Objectives describe the steps in gene cloning by using plasmid as the vector](https://reader035.vdocuments.site/reader035/viewer/2022062519/5697bfee1a28abf838cb98bf/html5/thumbnails/39.jpg)
Screening
i.e. detecting the bacteria containing rDNA
Blue-white screening
![Page 40: 1 Objectives describe the steps in gene cloning by using plasmid as the vector](https://reader035.vdocuments.site/reader035/viewer/2022062519/5697bfee1a28abf838cb98bf/html5/thumbnails/40.jpg)
40
Probing (Nucleic acid hybridization)
- to identify bacteria with recombinant plasmid containing gene of interest (target gene)
![Page 41: 1 Objectives describe the steps in gene cloning by using plasmid as the vector](https://reader035.vdocuments.site/reader035/viewer/2022062519/5697bfee1a28abf838cb98bf/html5/thumbnails/41.jpg)