Restriction Analysis of pDRK and pGRN Original Plasmids Hin dIII Hin dIII pGRN Hin dIII pDRK ara C...
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Transcript of Restriction Analysis of pDRK and pGRN Original Plasmids Hin dIII Hin dIII pGRN Hin dIII pDRK ara C...
![Page 1: Restriction Analysis of pDRK and pGRN Original Plasmids Hin dIII Hin dIII pGRN Hin dIII pDRK ara C amp r kan r gfp P BAD.](https://reader034.fdocuments.net/reader034/viewer/2022051622/5697bff31a28abf838cbc7e1/html5/thumbnails/1.jpg)
Restriction Analysis of pDRK and pGRNOriginal Plasmids
HindIII
HindIII
pGRN
HindIII
pDRK
ara C
ampr
kanr
gfpPBAD
![Page 2: Restriction Analysis of pDRK and pGRN Original Plasmids Hin dIII Hin dIII pGRN Hin dIII pDRK ara C amp r kan r gfp P BAD.](https://reader034.fdocuments.net/reader034/viewer/2022051622/5697bff31a28abf838cbc7e1/html5/thumbnails/2.jpg)
Restriction Analysis of pDRK and pGRNRestriction Fragments after digest with HindIII
gfp
ara C
PBAD ampr
kanr
![Page 3: Restriction Analysis of pDRK and pGRN Original Plasmids Hin dIII Hin dIII pGRN Hin dIII pDRK ara C amp r kan r gfp P BAD.](https://reader034.fdocuments.net/reader034/viewer/2022051622/5697bff31a28abf838cbc7e1/html5/thumbnails/3.jpg)
Ligation of pDRK/pGRN Restriction Fragments
DNA ligase
AA G C T T A
T T C G A A G C T
T C G A
DNA ligase
gfp
![Page 4: Restriction Analysis of pDRK and pGRN Original Plasmids Hin dIII Hin dIII pGRN Hin dIII pDRK ara C amp r kan r gfp P BAD.](https://reader034.fdocuments.net/reader034/viewer/2022051622/5697bff31a28abf838cbc7e1/html5/thumbnails/4.jpg)
Ligation of pDRK/pGRN Restriction FragmentsRecombinant Plasmid of Interest
rpGLO PBAD
ampr
ara C
HindIII
HindIII
gfp
![Page 5: Restriction Analysis of pDRK and pGRN Original Plasmids Hin dIII Hin dIII pGRN Hin dIII pDRK ara C amp r kan r gfp P BAD.](https://reader034.fdocuments.net/reader034/viewer/2022051622/5697bff31a28abf838cbc7e1/html5/thumbnails/5.jpg)
Preparing Competent Cells for Transformation
++
---
--
-- -
----
---
Lipid bilayerinner membrane
Lipid bilayerouter membrane
Peptidoglycan layer
++++
++
++
++++
++
++
++++ ++ ++
++
++++
adhesion zone
phospholipids
calcium ion
![Page 6: Restriction Analysis of pDRK and pGRN Original Plasmids Hin dIII Hin dIII pGRN Hin dIII pDRK ara C amp r kan r gfp P BAD.](https://reader034.fdocuments.net/reader034/viewer/2022051622/5697bff31a28abf838cbc7e1/html5/thumbnails/6.jpg)
++
---
--
--- -
----
---
Lipid bilayerinner membrane
Lipid bilayerouter membrane
Peptidoglycan layer
++
++
++++
++
++
++++
++
++
++++ ++ ++
++
++
++++
++
++
++
++++
++
++
++
++++
plasmid
adhesion zone
phospholipids
calcium ion
Transforming Escherichia coli With a Recombinant Plasmid
![Page 7: Restriction Analysis of pDRK and pGRN Original Plasmids Hin dIII Hin dIII pGRN Hin dIII pDRK ara C amp r kan r gfp P BAD.](https://reader034.fdocuments.net/reader034/viewer/2022051622/5697bff31a28abf838cbc7e1/html5/thumbnails/7.jpg)
GFP Expression
ara C gene
gfp gene
promoter
mRNA
transcription
translation
C protein
![Page 8: Restriction Analysis of pDRK and pGRN Original Plasmids Hin dIII Hin dIII pGRN Hin dIII pDRK ara C amp r kan r gfp P BAD.](https://reader034.fdocuments.net/reader034/viewer/2022051622/5697bff31a28abf838cbc7e1/html5/thumbnails/8.jpg)
GFP Expression
C protein prevents gfp transcription by causing a loop to form in the region of the gfp gene
ara C gene
gfp gene
promoter
![Page 9: Restriction Analysis of pDRK and pGRN Original Plasmids Hin dIII Hin dIII pGRN Hin dIII pDRK ara C amp r kan r gfp P BAD.](https://reader034.fdocuments.net/reader034/viewer/2022051622/5697bff31a28abf838cbc7e1/html5/thumbnails/9.jpg)
GFP Expression
ara C gene gfp gene
promoter
arabinose
mRNA
Arabinose – C protein complex prevents DNA looping and helps to align RNA polymerase on the promoter site.
transcription
translation
GFP
RNA polymerase
arabinose – C protein complex
C protein
![Page 10: Restriction Analysis of pDRK and pGRN Original Plasmids Hin dIII Hin dIII pGRN Hin dIII pDRK ara C amp r kan r gfp P BAD.](https://reader034.fdocuments.net/reader034/viewer/2022051622/5697bff31a28abf838cbc7e1/html5/thumbnails/10.jpg)
Transforming Escherichia coli With a Recombinant Plasmid
Recombinant plasmids
Competent cells
transformed cells
![Page 11: Restriction Analysis of pDRK and pGRN Original Plasmids Hin dIII Hin dIII pGRN Hin dIII pDRK ara C amp r kan r gfp P BAD.](https://reader034.fdocuments.net/reader034/viewer/2022051622/5697bff31a28abf838cbc7e1/html5/thumbnails/11.jpg)
Growth of transformed bacteria on various plates
P+ plates
P- plates
LB LB/amp LB/amp/ara
LB LB/amp
No growth
![Page 12: Restriction Analysis of pDRK and pGRN Original Plasmids Hin dIII Hin dIII pGRN Hin dIII pDRK ara C amp r kan r gfp P BAD.](https://reader034.fdocuments.net/reader034/viewer/2022051622/5697bff31a28abf838cbc7e1/html5/thumbnails/12.jpg)
GFP
![Page 13: Restriction Analysis of pDRK and pGRN Original Plasmids Hin dIII Hin dIII pGRN Hin dIII pDRK ara C amp r kan r gfp P BAD.](https://reader034.fdocuments.net/reader034/viewer/2022051622/5697bff31a28abf838cbc7e1/html5/thumbnails/13.jpg)
Ligation of pDRK/pGRN Restriction FragmentsRecombinant Plasmid of Interest
rpGLO PBAD
ampr
ara C
HindIII
HindIII
gfp
![Page 14: Restriction Analysis of pDRK and pGRN Original Plasmids Hin dIII Hin dIII pGRN Hin dIII pDRK ara C amp r kan r gfp P BAD.](https://reader034.fdocuments.net/reader034/viewer/2022051622/5697bff31a28abf838cbc7e1/html5/thumbnails/14.jpg)