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Cold Shock Vectors. Optimal Growth Acclimation Steady Low Temp Growth Stationary 37ºC Phase Adapted Cells Phase. CSPs. Growth Curve. Non-CSPs. CSPs. Non-CSPs. Induction of cold-shock proteins in Esherichia coli. 37 °C. 15. , 30 min.

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Optimal GrowthAcclimation Steady Low Temp Growth Stationary

37ºCPhase Adapted Cells Phase

CSPs

Growth Curve

Non-CSPs

CSPs

Non-CSPs


Induction of cold-shock proteins in Esherichia coli

37°C

15

, 30 min

15

°C

, 1 hr

°C


Protein Unfolding

▪ Protein Degradation

Cold shock

Heat shock

▪ RNA Stabilization

RNA Chaperones

Molecular Chaperones

▪ Ribosome Dysfunction

RbfA, CsdA

Proteases

▪ mRNA Degradation

PNPase


W11

The primary and tertiary structure of CspA

-4



-5

-2

-1

MSGKMTGIVKWFNADKGFGFITPDDGSKDVFVHFSAIQNDGYKSLDEGQKVSFTIESGAKGPAAGNVTSL

RNP2

RNP1

F12

Y42

F20

F53

F34

F18

H33

F31


The molecule number of CspA homologues

in cold-shocked cells

Cold shock time (hr)

Purified CspA protein (ng)

0 1 3 6

1 2 4 8 16 32 64 128

37ºC

15ºC

C

I

C

I

E

B

A G

E

B

A G

CspA : 1 × 106 molecules/cell

( CspA + B + G + E : 2 × 106 molecules/cell )


Effect of nonsense mutations

AUG

A

cspA promotor 5’-UTR SD coding sequence 3’-UTR

*

pJJG02

*

*

pA01S

*

*

pA10S

*

*

pA30S

pUC19 pJJG02 pA01S pA10S pA30S

B

C

5

0 1 3 6 hr

0 1 3 6

0 1 3 6

CS: 0 1 3 6

01 3 6

pUC19

4.5

pJJG02

4

pA01S

pA10S

3.5

pA30S

3

OD600

2.5

2

1.5

1

0.5

0

0

2

4

6

8

10

12

14

16

18

20

22

24

26

28

30

32

Time after cold shock (hr)



LACE

LowTemperature-dependentAntibiotic Effect

of Truncated cspAExpression

Ribosome Trapping


Cold-Shock Vector

Single-Protein Synthesis

at low temperature

without producing any other

cellular proteins

NMR without protein purification


cspA 3’-UTR

BamHI

KpnI

HindIII

NdeI

SalI

Multiple

cloning site

Factor Xa cleavage site

[His]6

cspA ORF

Ap

pCold

(prototype)

3100bp

cspA 5’-UTR

T7/cspA promoter

ori


Induction at 15 oC

with pCold I

Induction at 37 oC

with a T7 vector (3 hr)

_______ ______ ______ _______ _______

0 12 24 0 12 24 0 12 24 0 12 24 0 12 24 hr

- + - + - + - + - +

____ ____ ____ _____ _____

WR:49 35 26 27 53

WR49 WR35 WR26 WR27 WR53

Production of C. elegans Proteins


-216 kDa

-132

-45.7

-32.5

-18.4

_________ __________ _________

0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 5 days

WR49 WR35 WR26


Expression of γINF in pcold II vector

-interferon

0 1 3 6 hr

at 15° C

1 mM IPTG


A

B

cspA 3’-UTR

cspA 3’-UTR

BamHI

BamHI

NdeI

NdeI

Factor Xa cleavage site

DB

[His]6

cspA 5’-UTR

cspA ORF

Ap

pCold I

(3100bp)

Ap

pCold II

(2900bp)

cspA promoter

cspA 5’-UTR

cspA promoter

ori

ori

C

cspA 3’-UTR

BamHI

NdeI

cspA 5’-UTR

Ap

pCold III

(2900bp)

cspA promoter

ori


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