gene transfer techniques · ti-plasmid mediated transfer of gene into a plant • the ti-plasmid...

GENE TRANSFER IN BACTERIAby

unit 8

GENE TRANSFER IN BACTERIAby

unit 8

GENE TRANSFER IN BACTERIAby

unit 8

GENE TRANSFER IN BACTERIAby

unit 8Subject: Life sciences

Faculty: Ramesh KumarNew Life College of Nursing

Date 08/01/2016

Subject: Life sciences

Faculty: Ramesh KumarNew Life College of Nursing

Date 08/01/2016

GENE TRANSFER IN BACTERIAby

unit 8

GENE TRANSFER IN BACTERIAby

unit 8

GENE TRANSFER IN BACTERIAby

unit 8

GENE TRANSFER IN BACTERIAby

unit 8Subject: Life sciences

Faculty: Ramesh KumarNew Life College of Nursing

Date 08/01/2016

Subject: Life sciences

Faculty: Ramesh KumarNew Life College of Nursing

Date 08/01/2016

METHODS OF GENE TRANSFERMETHODS OF GENE TRANSFER

DNA transfer by natural methods• 1. Conjugation

• 2. Bacterial transformation

• 3. Retroviral transduction

• 4. Agrobacterium mediated transfer

DNA transfer by natural methods• 1. Conjugation

• 2. Bacterial transformation

• 3. Retroviral transduction

• 4. Agrobacterium mediated transfer

METHODS OF GENE TRANSFERMETHODS OF GENE TRANSFER

DNA transfer by natural methods• 1. Conjugation

• 2. Bacterial transformation

• 3. Retroviral transduction

• 4. Agrobacterium mediated transfer

DNA transfer by natural methods• 1. Conjugation

• 2. Bacterial transformation

• 3. Retroviral transduction

• 4. Agrobacterium mediated transfer

CONJUGATIONCONJUGATION

• Requires the presence of a special plasmid called the Fplasmid.

• Bacteria that have a F plasmid are referred to as as F+ ormale. Those that do not have an F plasmid are F- of female.

• The F plasmid consists of 25 genes that mostly code forproduction of sex pilli.

• A conjugation event occurs when the male cell extends hissex pilli and one attaches to the female.

• Requires the presence of a special plasmid called the Fplasmid.

• Bacteria that have a F plasmid are referred to as as F+ ormale. Those that do not have an F plasmid are F- of female.

• The F plasmid consists of 25 genes that mostly code forproduction of sex pilli.

• A conjugation event occurs when the male cell extends hissex pilli and one attaches to the female.

CONJUGATIONCONJUGATION

• Requires the presence of a special plasmid called the Fplasmid.

• Bacteria that have a F plasmid are referred to as as F+ ormale. Those that do not have an F plasmid are F- of female.

• The F plasmid consists of 25 genes that mostly code forproduction of sex pilli.

• A conjugation event occurs when the male cell extends hissex pilli and one attaches to the female.

• Requires the presence of a special plasmid called the Fplasmid.

• Bacteria that have a F plasmid are referred to as as F+ ormale. Those that do not have an F plasmid are F- of female.

• The F plasmid consists of 25 genes that mostly code forproduction of sex pilli.

• A conjugation event occurs when the male cell extends hissex pilli and one attaches to the female.

• This attached pilus is a temporary cytoplasmic bridgethrough which a replicating F plasmid is transferred fromthe male to the female.

• When transfer is complete, the result is two male cells.

• When the F+ plasmid is integrated within the bacterialchromosome, the cell is called an Hfr cell (high frequencyof recombination cell).

• This attached pilus is a temporary cytoplasmic bridgethrough which a replicating F plasmid is transferred fromthe male to the female.

• When transfer is complete, the result is two male cells.

• When the F+ plasmid is integrated within the bacterialchromosome, the cell is called an Hfr cell (high frequencyof recombination cell).

• This attached pilus is a temporary cytoplasmic bridgethrough which a replicating F plasmid is transferred fromthe male to the female.

• When transfer is complete, the result is two male cells.

• When the F+ plasmid is integrated within the bacterialchromosome, the cell is called an Hfr cell (high frequencyof recombination cell).

• This attached pilus is a temporary cytoplasmic bridgethrough which a replicating F plasmid is transferred fromthe male to the female.

• When transfer is complete, the result is two male cells.

• When the F+ plasmid is integrated within the bacterialchromosome, the cell is called an Hfr cell (high frequencyof recombination cell).

TRANSFORMATIONTRANSFORMATION

• transformation is the direct uptake of exogenous DNA from itssurroundings and taken up through the cell membrane .

• Transformation occurs naturally in some species of bacteria,but it can also be effected by artificial treatment in otherspecies.

• Cells that have undergone this treatment are said to becompetent.

• Any DNA that is not integrated into he chromosome will bedegraded.

• transformation is the direct uptake of exogenous DNA from itssurroundings and taken up through the cell membrane .

• Transformation occurs naturally in some species of bacteria,but it can also be effected by artificial treatment in otherspecies.

• Cells that have undergone this treatment are said to becompetent.

• Any DNA that is not integrated into he chromosome will bedegraded.

TRANSFORMATIONTRANSFORMATION

• transformation is the direct uptake of exogenous DNA from itssurroundings and taken up through the cell membrane .

• Transformation occurs naturally in some species of bacteria,but it can also be effected by artificial treatment in otherspecies.

• Cells that have undergone this treatment are said to becompetent.

• Any DNA that is not integrated into he chromosome will bedegraded.

• transformation is the direct uptake of exogenous DNA from itssurroundings and taken up through the cell membrane .

• Transformation occurs naturally in some species of bacteria,but it can also be effected by artificial treatment in otherspecies.

• Cells that have undergone this treatment are said to becompetent.

• Any DNA that is not integrated into he chromosome will bedegraded.

THE Ti-PLASMIDSTHE Ti-PLASMIDS• A remarkable feature of the Ti plasmid is that, after infection, part of

the molecule is integrated into the plant chromosomal DNA .

• This segment, called the T-DNA, is between 15 and 30 kb in size,depending on the strain.

• T-DNA contains eight or so genes that are expressed in the plant celland are responsible for the cancerous properties of the transformedcells.

• These genes also direct synthesis of unusual compounds, calledopines, that the bacteria use as nutrient.

• A remarkable feature of the Ti plasmid is that, after infection, part ofthe molecule is integrated into the plant chromosomal DNA .

• This segment, called the T-DNA, is between 15 and 30 kb in size,depending on the strain.

• T-DNA contains eight or so genes that are expressed in the plant celland are responsible for the cancerous properties of the transformedcells.

• These genes also direct synthesis of unusual compounds, calledopines, that the bacteria use as nutrient.

THE Ti-PLASMIDSTHE Ti-PLASMIDS• A remarkable feature of the Ti plasmid is that, after infection, part of

the molecule is integrated into the plant chromosomal DNA .

• This segment, called the T-DNA, is between 15 and 30 kb in size,depending on the strain.

• T-DNA contains eight or so genes that are expressed in the plant celland are responsible for the cancerous properties of the transformedcells.

• These genes also direct synthesis of unusual compounds, calledopines, that the bacteria use as nutrient.

• A remarkable feature of the Ti plasmid is that, after infection, part ofthe molecule is integrated into the plant chromosomal DNA .

• This segment, called the T-DNA, is between 15 and 30 kb in size,depending on the strain.

• T-DNA contains eight or so genes that are expressed in the plant celland are responsible for the cancerous properties of the transformedcells.

• These genes also direct synthesis of unusual compounds, calledopines, that the bacteria use as nutrient.

• The vir (virulence) region of the Ti- plasmid contains thegenes required for the T-DNA transfer process.

• The genes in this region encode the DNA processingenzymes required for excision, transfer and integration of theT-DNA segment.

• The vir (virulence) region of the Ti- plasmid contains thegenes required for the T-DNA transfer process.

• The genes in this region encode the DNA processingenzymes required for excision, transfer and integration of theT-DNA segment.

• The vir (virulence) region of the Ti- plasmid contains thegenes required for the T-DNA transfer process.

• The genes in this region encode the DNA processingenzymes required for excision, transfer and integration of theT-DNA segment.

• The vir (virulence) region of the Ti- plasmid contains thegenes required for the T-DNA transfer process.

• The genes in this region encode the DNA processingenzymes required for excision, transfer and integration of theT-DNA segment.

Ti-Plasmid mediatedtransfer of gene into a plant

Ti-Plasmid mediatedtransfer of gene into a plant

• The Ti-Plasmid has an innate ability to transmit bacterial DNAinto plant cells.

• The gene of a donor organism can be introduced into the Tiplasmid at the T-DNA region

• This plasmid now becomes a recombinant plasmid.• By Agrobacterium infection, the donor genes can transferred

from the recombinant Ti- Plasmid and integrated into thegenotype of the host plant.

• The Ti-Plasmid has an innate ability to transmit bacterial DNAinto plant cells.

• The gene of a donor organism can be introduced into the Tiplasmid at the T-DNA region

• This plasmid now becomes a recombinant plasmid.• By Agrobacterium infection, the donor genes can transferred

from the recombinant Ti- Plasmid and integrated into thegenotype of the host plant.

Ti-Plasmid mediatedtransfer of gene into a plant

Ti-Plasmid mediatedtransfer of gene into a plant

• The Ti-Plasmid has an innate ability to transmit bacterial DNAinto plant cells.

• The gene of a donor organism can be introduced into the Tiplasmid at the T-DNA region

• This plasmid now becomes a recombinant plasmid.• By Agrobacterium infection, the donor genes can transferred

from the recombinant Ti- Plasmid and integrated into thegenotype of the host plant.

• The Ti-Plasmid has an innate ability to transmit bacterial DNAinto plant cells.

• The gene of a donor organism can be introduced into the Tiplasmid at the T-DNA region

• This plasmid now becomes a recombinant plasmid.• By Agrobacterium infection, the donor genes can transferred

from the recombinant Ti- Plasmid and integrated into thegenotype of the host plant.

ConclusionConclusion

Transformation

Conjugation

Gene transfer in bacteria

Transformation

Conjugation

Gene transfer in bacteria

ConclusionConclusion

REFERENCESREFERENCES• Sukharev, S.I., Klenchin, V.A., Serov, S.M., Chernomordik, L.V. & Chizmadzhev, Y.A. (1992). Electroporation• and electrophoretic DNA transfer into cells. The effect of DNA interaction with electropores. Biophys. J., 63 (5):• 1320-1327.• [2] Chu, G., Hayakawa, H. & Berg, P. (1987). Electroporation for the efficient transfection of mammalian cells with• DNA. Nucleic Acids Res., 15 (3): 1311-1326.• [3] Akamatsu, W., Okano, H.J., Osumi, N., Inoue, T., Nakamura, S., Sakakibara, S.I., Miura, M., Matsuo, N.,

Darnell,• R.B. & Okano, H. (1999). Mammalian ELAV-like neuronal RNAbinding proteins HuB and HuC promote• neuronal development in both the central and the peripheral nervous systems. Proc. Natl. Acad. Sci. USA., 96

(17):• 9885-9890.• [4] Osumi, N. & Inoue, T. (2001). Gene transfer into cultured mammalian embryos by electroporation. Methods., 24• Antoni Ivorra, Boris Rubinsky. "Gels with predetermined conductivity used in electroporation of tissue USPTO

Application #: 20080214986 - Class: 604 21 (USPTO)".• Jump upJump up^ Sugar, I.P.; Neumann, E. (1984). "Stochastic model for electric field-induced membrane pores

electroporation". Biophysical Chemistry 19 (3): 211–25. doi:10.1016/0301-4622(84)87003-9. PMID 6722274.• ^ Alberts, Bruce; et al. (2002). Molecular Biology of the Cell. New York: Garland Science. p. G:35. ISBN 978-0-81

• Sukharev, S.I., Klenchin, V.A., Serov, S.M., Chernomordik, L.V. & Chizmadzhev, Y.A. (1992). Electroporation• and electrophoretic DNA transfer into cells. The effect of DNA interaction with electropores. Biophys. J., 63 (5):• 1320-1327.• [2] Chu, G., Hayakawa, H. & Berg, P. (1987). Electroporation for the efficient transfection of mammalian cells with• DNA. Nucleic Acids Res., 15 (3): 1311-1326.• [3] Akamatsu, W., Okano, H.J., Osumi, N., Inoue, T., Nakamura, S., Sakakibara, S.I., Miura, M., Matsuo, N.,

Darnell,• R.B. & Okano, H. (1999). Mammalian ELAV-like neuronal RNAbinding proteins HuB and HuC promote• neuronal development in both the central and the peripheral nervous systems. Proc. Natl. Acad. Sci. USA., 96

(17):• 9885-9890.• [4] Osumi, N. & Inoue, T. (2001). Gene transfer into cultured mammalian embryos by electroporation. Methods., 24• Antoni Ivorra, Boris Rubinsky. "Gels with predetermined conductivity used in electroporation of tissue USPTO

Application #: 20080214986 - Class: 604 21 (USPTO)".• Jump upJump up^ Sugar, I.P.; Neumann, E. (1984). "Stochastic model for electric field-induced membrane pores

electroporation". Biophysical Chemistry 19 (3): 211–25. doi:10.1016/0301-4622(84)87003-9. PMID 6722274.• ^ Alberts, Bruce; et al. (2002). Molecular Biology of the Cell. New York: Garland Science. p. G:35. ISBN 978-0-81

REFERENCESREFERENCES• Sukharev, S.I., Klenchin, V.A., Serov, S.M., Chernomordik, L.V. & Chizmadzhev, Y.A. (1992). Electroporation• and electrophoretic DNA transfer into cells. The effect of DNA interaction with electropores. Biophys. J., 63 (5):• 1320-1327.• [2] Chu, G., Hayakawa, H. & Berg, P. (1987). Electroporation for the efficient transfection of mammalian cells with• DNA. Nucleic Acids Res., 15 (3): 1311-1326.• [3] Akamatsu, W., Okano, H.J., Osumi, N., Inoue, T., Nakamura, S., Sakakibara, S.I., Miura, M., Matsuo, N.,

Darnell,• R.B. & Okano, H. (1999). Mammalian ELAV-like neuronal RNAbinding proteins HuB and HuC promote• neuronal development in both the central and the peripheral nervous systems. Proc. Natl. Acad. Sci. USA., 96

(17):• 9885-9890.• [4] Osumi, N. & Inoue, T. (2001). Gene transfer into cultured mammalian embryos by electroporation. Methods., 24• Antoni Ivorra, Boris Rubinsky. "Gels with predetermined conductivity used in electroporation of tissue USPTO

Application #: 20080214986 - Class: 604 21 (USPTO)".• Jump upJump up^ Sugar, I.P.; Neumann, E. (1984). "Stochastic model for electric field-induced membrane pores

electroporation". Biophysical Chemistry 19 (3): 211–25. doi:10.1016/0301-4622(84)87003-9. PMID 6722274.• ^ Alberts, Bruce; et al. (2002). Molecular Biology of the Cell. New York: Garland Science. p. G:35. ISBN 978-0-81

• Sukharev, S.I., Klenchin, V.A., Serov, S.M., Chernomordik, L.V. & Chizmadzhev, Y.A. (1992). Electroporation• and electrophoretic DNA transfer into cells. The effect of DNA interaction with electropores. Biophys. J., 63 (5):• 1320-1327.• [2] Chu, G., Hayakawa, H. & Berg, P. (1987). Electroporation for the efficient transfection of mammalian cells with• DNA. Nucleic Acids Res., 15 (3): 1311-1326.• [3] Akamatsu, W., Okano, H.J., Osumi, N., Inoue, T., Nakamura, S., Sakakibara, S.I., Miura, M., Matsuo, N.,

Darnell,• R.B. & Okano, H. (1999). Mammalian ELAV-like neuronal RNAbinding proteins HuB and HuC promote• neuronal development in both the central and the peripheral nervous systems. Proc. Natl. Acad. Sci. USA., 96

(17):• 9885-9890.• [4] Osumi, N. & Inoue, T. (2001). Gene transfer into cultured mammalian embryos by electroporation. Methods., 24• Antoni Ivorra, Boris Rubinsky. "Gels with predetermined conductivity used in electroporation of tissue USPTO

Application #: 20080214986 - Class: 604 21 (USPTO)".• Jump upJump up^ Sugar, I.P.; Neumann, E. (1984). "Stochastic model for electric field-induced membrane pores

electroporation". Biophysical Chemistry 19 (3): 211–25. doi:10.1016/0301-4622(84)87003-9. PMID 6722274.• ^ Alberts, Bruce; et al. (2002). Molecular Biology of the Cell. New York: Garland Science. p. G:35. ISBN 978-0-81

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