1. Chapter 6 Manipulation of Gene Expression in Prokaryotes

2. Molecular Biological Features for the Manipulation of Gene Expression
Promoter and Terminator
Ribosome binding site
Copy number and Genetic stability
Final location of the foreign protein
Translational efficiency in the host organism
Stability of the cloned gene protein

3. Host Cell E. coli Bacillus subtilis Yeast Animal cell Plant cell
most well-known in its genetics, molecular biology, biochemistry, and physiology
Bacillus subtilis
Yeast
Animal cell
Plant cell
Insect cell

4. Gene Expression from Strong and Regulatable Promoters
Strong promoter
RNA polymerase recognition and/or binding is strong.
Transcription frequency is high.
 more mRNA
The sequence of the -35 and -10 regions are important for the strength of promoter.

5. Gene Expression from Strong and Regulatable Promoters
Constitutive expression of foreign protein
Can be detrimental to the host because of the energy drain
Can cause plasmid instability
Regulatable promoters
control the rate of transcription
E. coli lac, trp, tac (-10 lac trp) promoter
Bacteriophage l pL promoter
Bacteriophage T7 gene10 promoter

6. lac promoter
Induced by lactose or IPTG (isoproply-b-D-thiogalactopyranoside)
Repressed by glucose (catabolic repression)
Glucose (↓)  cAMP (↑)
cAMP (↑)  CAP-Promoter affinity (↑)
CAP (catabolite activator protein) enhances the binding of RNA polymerase.
lacUV5
a variant of lac promoter,
stronger than wild-type lac promoter

7. Lac Promoter

8. trp promoter
Derepressed by removing tryptophan or by adding 3-indoleacrylic acid (IAA)
trp repressor
Binding to the operator in the presence of tryptophan
Leaky expression

9. tac promoter -10 region of lac promoter and -35 region of trp promoter
Induced by lactose or IPTG
Strength: tac > trp > lac (10: 3.3: 1)

10. Bacteriophage l PL Promoter
Induced by shifting temperature from ~30oC to ~42oC
Repressor : cI
cI857: temperature-sensitive cI
Active at low temperature (28~30oC)
Inactive at high temperature (42oC)

11. Bacteriophage l PL Promoter

12. T7 gene 10 promoter (pT7)
from bacteriophage T7 gene 10
Induced by IPTG
pET vectors

13. T7 gene 10 promoter T7 promoter T7 RNA polymerase gene
Transcription by T7 RNA polymerase
T7 RNA polymerase gene
In the E. coli chromosome on a bacteriophage l lysogen
Under the control of the lac promoter
Induced by IPTG

14. Increasing Protein Production
* pKN * pPLc2833
~ effective ori ~ strong promoter (pL) and
high copy number multiple cloning site (MCS)
(28oC: 82  42oC: 521) (fragment 1)
(fragment c) ~ selectable marker (Ampr)
(fragment 3) ↓↓
pCP3 (fragments 1, 3, and c)

15. pCP3

16. Large-Scale System Temp. shift Chemical inducer
time & energy
Chemical inducer
high cost
Inexpensive system is necessary for large scale protein production

17. Large-Scale System Two plasmid system (Figure 6.5)
cI under the control of trp promoter
Protein expression under the control of pL promoter
molasses and casein hydrolysate (No tryptophan)
 then, addition of trypton (containing tryptophan)
Control gene expression
Without Trp
Repression of protein expression
With Trp
Induction of protein expression

18. Dual Plasmid System

19. Expression in Other Microorganisms
The best promoter for use in a particular organism is not necessarily the one that is most efficient in E. coli (Table 6.2).
Tn5 (70-bp DNA fragment from one of the terminal inverted repeats of transposon 5) functions in a wide range of bacterial hosts.
Tn5 promoters can be used in a number of different bacterial host.
Universal gram (-) bacterial expression vector
Tn5 promoter in a broad-host-range plasmid pRK290
Efficient gene expression in different bacterial hosts