regulation studies of argo of e.coli by lysg mutants of corynebacterium glutamicum

7/29/2019 Regulation studies of argO of E.coli by LysG mutants of Corynebacterium glutamicum

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La

Centre fo

Regulatio

LysG mu

Sum

Indian

Indian NatiThe Nationa

1

boratory of Bacterial Genetics

DNA Fingerprinting and Diagnos

Guide: Dr. . Gowrishankar

studies ofargO of

tants ofCorynebact

glutamicum

er Research Fellowship 20

by

Aayudh Das

University of Calcutta

Academy of Sciences, Bangalore

onal Academy of Sciences, New DAcademy of Sciences India, Allah

tics

.coliby

rium

2

lhiabad


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2

ACKNOWLEDGEMENT

I desire to show my sense of gratitude to the Indian Academy of Sciences, Indian National Academy of

Sciences and National Academy of Sciences for granting me the Summer Research Fellowship to work in

the Laboratory of Bacterial Genetics, CDFD Hyderabad.

My sincere thanks to Dr. J. Gowrishankar , Director, CDFD and Head, Laboratory of Bacterial Genetics as

well as my guide for permitting me to work in his laboratory as a summer trainee and allowing to attend

his lab meetings and seminars.

I thank Dr. K. Anupama, Scientist III, CDFD and my supervisor, for her patient guidance and support.

I acknowledge gratefully all LBGians, Amit, Nora, Aanisa, Amar, Raj, Shanthy, Mishraji, Suchitra, Shaffiqu,

Dr. Krishna Leela, Dr. Bindu, Dr. Vimala, for helping me out in different occasions. Dr. Harinarayanan and

Dr. Abhijit deserve special mention for useful discussions and criticism.

I wish to thank Vamsee, Giri and Debashish for providing me with media and other chemicals during my

work at LBG.

I thank NGTF for providing me with the DNA sequences on time.

To my friends and well-wishers of other laboratories and my co-summer trainees, my gratitude is

boundless- they made my stay in CDFD immensely enjoyable.

I have received complete support from my parents and sister all through and this has been a great source

of strength.

Aayudh Das

27.07.2012


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3

~CONTENT~

ABSTRACT INTRODUCTION

OBJECTIVE

Materials

METHODS

RESULTS

DISCUSSION

REFERENCE


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4

ABSTRACT

The pairs ArgP-argO of Escherichia coli and LysG-lysE of Corynebacterium glutamicum are orthologous,

with the first member of each pair being a LysR-type transcriptional regulator and the second its target

encoding a basic amino acid exporter. Whereas LysE is an exporter of arginine (Arg) and lysine (Lys)

whose expression is induced by Arg, Lys, or histidine (His), ArgO exports Arg alone and its expression is

activated by Arg but not Lys or His. Of several ArgP-dominant (ArgPd) variants that confer elevated Arg-

independent argO expression, ArgP (S94L), ArgP (P108S) and ArgP (P274S) activates lysEexpression in E.

coli. We constructed several LysG mutants at the corresponding residues of LysG namely T94L, P108S and

L274S to determine if they regulate argO. In the presence of arginine all those mutants activates the

transcription ofargO. So we can conclude that LysG mutants regulate argO in the presence of arginine.


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INTRODUCTIO

A common mechanism for activatio

transcription factor of RNA polym

productive transcription.

LTTRs-

The family ofLysR-type transcripti

and Gram-negative bacteria, with

These proteins are involved in mod

cases is achieved by their binding t

between an individual protein, its

cross-talk has been identified bet

related function such as those for

reported between LTTR ortholo

transcriptional regulators share co

the mechanisms by which they bo

extensive structural homology espe

(A)

RBS

(B)

RBS

Fig 1: Schematic presentation (A)

transcription as RNAP is not rec

transcription as RNAP gets recruite

5

N

n of gene expression in all organisms is that

erase (RNAP) to a promoter so that the l

nal regulators (LTTRs) is widely distributed

multiple paralogs being represented even

ulating an extremely diverse set of metabol

co-effector ligands. Considerable specificit

co-effector(s) and cognate target(s); nevert

een LysR-type paralogs in a single bacteri

catabolism of aromatic compounds but no

s. Structural studies have indicated tha

mon protein folds, they differ in their oligo

th bind to DNA regulatory regions and co

cially at N-terminal DNA binding domain.

ABS -35 -10

Active

ABS -35 -10 +1

showing that LTTRs in absence of any c

ruited. (B) LTTRs in the presence any c

d.

involving recruitment by a

atter can then engage in

across both Gram-positive

within a single organism.

ic functions which in most

y exists in the interactions

heless, a limited extent of

ium that control genes of

cross regulation has been

although the LysR-type

merization properties and

tact RNAP. They have an

1

transcription

o-effectors cannot induce

-effectors is able induce


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LysG-LysE ofC. glutamic

The proteins LysG and ArgP are

positive Corynebacterium glutam

regulator of LysE expression and liLysE and ArgO belongs to the famil

Fig 2: Schematic representation sh

LysE exports both lysine (Lys) and

transcription in presence of Arg, L

not Lys or His;

(A)

(i)

(ii)

(iii)

Fig 3: Schematic presentation (

transcription ofargOas RNAP is n

expression takes place. iii- In the

6

um and ArgP-argO of E.coli-

orthologous members of the LysR family

cum and Gram-negative Escherichia coli.

ikewise ArgP regulates expression of ArgO of amino acid exporters in bacteria.

wing LysG regulates LysE and ArgPregula

rginine (Arg), ArgO is an exporter only of A

s or histidine (His), while ArgP activates ar

(B)

(i)

(ii)

A) i- showing that ArgP without any c

t recruited. ii- In the presence of arginine R

presence of lysine ArgP actively shuts off

from, respectively, Gram-

LysG is a transcriptional

which is a LysE ortholog.

tes ArgO.

rg. The LysG activates lysE

gO in presence of Arg but

o-effector cannot induce

NAP is recruited and argO

argO transcription. (B) i-


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showing that LysG without any co-

ii- In the presence of arginine or lys

Cross regulation betwee

Fig 4: Schematic presentation show

LysE regulate argOnor ArgP regula

Sequence Al ignment stud

Sequence alignment ClustalW wa

observed the corresponding residu

LysG were T94L, P108S, L274S.

Fig 5: Schematic representationglutamicum.

7

effector cannot induce transcription of LysE

ine RNAP gets recruited and LysE transcript

LTTRs-

1. It has been repo

regulate argO

2. ArgP doesnt regulat

But is has been reporte

mutants namely S94L a

lysE constitutively but

R217L are unable to acti

ing neither

te lysE.

ies ofArgP andLysG-

used to obtain ArgP of E.coli and LysG

es for E.coliArgP (T94L) , ArgP (L274S), Arg

howing the sequence alignment of ArgP

as RNAP is not recruited.

ion takes place.

rted that LysG doesnt

e lysE

d that some of the ArgPd

nd P274S, P108S activate

V144M, Q65V, R295L,

vate lysE.

of C. glutamicum. It was

(P108S) in C. glutamicum

of E.coli and LysG of C.


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8

Table 1: LysG mutants-

LysG mutants Amino acid change

L274S Lysine to Serine (CTGTCT/AGA)

T94L Theronine to Lysine (ACCTTA/AAT)

P108S Proline to Serine (CCCTCT/AGA)

V144M Valine to Methionine (GTAATG/TAC)

Q65V Glutamine to Valine (CAAGTG/CAC)

R295C Arginine to Cysteine (CGGTCT/ACA)

R217L Arginine to Lysine (CGCTTA/AAT)

OBJECTIVE

The study aims at finding the effect of LysG mutants (T94L, L274S, P108S, V144M, Q65V, R295L, R217L) on

argO expression in E. coli.

MATERIALS

Bacterial strains: All the bacterial strains that were used in this study were derivatives of Escherichia coli

K-12 and their genotypes have been listed below.

Table 2: List of Bacterial strains-

Strains Genotypes

MC4100 F-araD139(argF-lac) U169rpsL150rela1 F16B5301 fruA25 deeoC1 ptsF25 e14

DH5 fhuA2 (argF-lacZ) U169 phoA glnV44 80 (lacZ)M15 gyrA96 recA1 relAl endA1 thi-

1 hsdR17


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9

Table 3: List of primers-

Name Sequence (5-3) Tm (C) DescriptionJGHlysGL274SF TGTATTGGCAACGATGGCGCTCTGAATCTAGATC

TCTAGCTAG

60 23bp, used for

L274S mutant

JGHlysGL274SR CTAGCTAGAGATCTAGATTCAGAGCGCCATCGTT

GCCAATACA

60 23bp, used for

L274S mutant

JGHlysGP108SF CGCTATCCACATGGTTTCCTTCTGTGTTCAACGA

GGTAGCTTC

61 23bp, used for

P108S mutant

JGHlysGP108SR GAAGCTACCTCGTTGAACACAGAAGGAAACCATG

TGGATAGCG

61 23bp, used for

P108S mutant

JGHlysGT94LF GCCTTGCTGAAATCCCGTTATTAATCGCCATCAA

CGCAGATTC

61 23bp, used for

T94L mutant

JGHlysGT94LR GAATCTGCGTTGATGGCGATTAATAACGGGATTT

CAGCAAGGC

61 23bp, used for

T94L mutant

JGHlysGP108SF CGCTATCCACATGGTTTCCTTCTGTGTTCAACGA

GGTAGCTTC

61 23bp, used for

P108S mutant

JGHlysGP108SR GAAGCTACCTCGTTGAACACAGAAGGAAACCATG

TGGATAGCG

61 23bp, used for

P108S mutant

JGHlysGV144MF GTGGAGATGTTTTAGGAGCGATGACCCGTGAAGC

TAATCCCGT

63 23bp, used for

V144M mutant

JGHlysGV144MR ACGGGATTAGCTTCACGGGTCATCGCTCCTAAAA

CATCTCCAC

63 23bp, used for

V144M mutant

JGHlysGR217LF ATGGTCCTGTGGGGCGCAGGTTAGTATCCATTGT

CCCGTCGGC

69 23bp, used for

R217L mutant

JGHlysGR217LR GCCGACGGGACAATGGATACTAACCTGCGCCCCA

CAGGACCAT

69 23bp, used for

R217L mutant

JGHlysGR295CF ATGCAGCAATCGAGGGATTGTGTCCTTAGTTACT

TCTGAAAAG

58 23bp, used for

R295C mutant

JGHlysGR295CR CTTTTCAGAAGTAACTAAGGACACAATCCCTCGA

TTGCTGCAT

58 23bp, used for

R295C mutant


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Table 4: Plasmids-

Plasmid

pHYD2676 It is a

pHYD1723 I

pHYD2677 It is a pMU575 d

pHYD2606

pHYD929

pBAD18 pMB9 rep

pCL1920

Vector maps-

10

Description

pBAD18 construct of LysG cloned at EcoR

t is a pMU575 construct ofargO regulator

rivative carryin, a 334-bp PstI-BamHI fra

region.

It is a pCL1920 derivative of argP (p27

pCL1920 derivative of argP (P217L

licon for Ara-induced expression of target

SC101 replicon, streptomycin and spectin

and HindIII

region

ment oflysEregulatory

4S)

genes, ampicillin

omycin


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11

Media and Buffers:

All the media and buffers were sterilized by autoclaving for 15 minutes at 121C under 15 psi pressure.

Media and buffers used in this study are as described below.

LB medium:

Tryptone : 10 g

Yeast extract : 5 g

NaCl : 10 g

Water to : 1000 ml

pH adjusted to 7.0-7.2 with 1N NaOH.

LB Agar:

LB medium : 1000 ml

Bacto-agar : 15 g

LB MES Agar (pH 5.8):

MES : 3.904g

Bacto tryptone: 2g

NaCl : 2g

Yeast extract : 1g

Bacto-agar : 30g

Made up the volume to 200 ml using autoclaved water

Minimal Agar (0.1 M; pH 5.8):

KHPO : 0.017M


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12

KHPO : 0.183 M

(NH)SO : 0.2 %

Sodium Citrate: 0.1 %

Made up to 100ml using autoclaved water

TAE buffer:

40 mM Tris-acetate, 2 mM EDTA (pH 8.0)

It was used as standard electrophoresis buffer. It was prepared as 50X concentrated stock solutions and

used at 1X concentration.

Chemicals-

No. Chemical Final Concentration

1. Glucose 0.2 %

2. Arabinose 0.2 %

3. CaCl2 0.1M

4. Glycerol 0.8%

5. Thiamine ( B ) 0.0001 %

6. MgSO 0.001M

7. Lysine 10mM

8. Arginine 10mM

9. X-gal 80g/ml

10. IPTG 100M

11. ONPG 4mg/ml

12.SDS 0.1%


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13

An tib io ti cs -

Antibiotics

Working Concentration (in g/ml)

Abbreviated in text as

LB MA

Spectinomycin 50 50 Spec

Trimethoprim 60 30 TP

Streptomycin 100 100 Strep

Ampicilin 40 40 Amp


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METHODS

Isolation of plasmid DN

For high purity plasmid RBC HiYield

Restriction Digestion-

0.5-1g of DNA was used for restri

appropriate 10X buffers supplied b

reaction volume of 25l. The diges

fragments were visualized by ethidi

Commercially available DNA size

with the digestion samples to comp

Reaction mixture-

1) PCR product-20.5 l, EcoRI-

2) pBAD18 plasmid-20.5 l, Ec

Ligation-

100-200ng of DNA was used in ea

The reaction was done in 10l vol

0.5-1 units of T4 DNA ligase. The re

Reaction mixture-

14

MOLECULAR TECHNIQUES:

-

Plasmid Mini Kit were used where ever re

ction enzyme digestion. 5-20 units of the r

y the manufacturers at final concentration

tion was allowed to proceed for a minimu

um bromide staining following electrophore

arkers (GeneRuler) of 1kb and HindIII la

are with and estimate the sizes of the restri

l, HindIII-1l, Digestion buffer-2.5l. Total

RI-1l, HindIII-1l, Digestion buffer-2.5l. T

Fig 6:

showi

and d

h ligation reaction. The vector to insert ra

umes containing ligation buffer (provided b

ction was carried at 21C for 16h (overnigh

uired.

striction enzyme with the

of 1X were used in a total

of 6h at 37C. The DNA

sis on 0.8-1% agarose gels.

mba DNA were run along

tion fragments.

= 25l

otal = 25l

Schematic representation

ng digested PCR product

igested pBAD18 plasmid.

io was maintained at 1:3.

y the manufacturers) and

).


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pBAD18 digested purified-2l, PC

ATP-0.5ul, Total reaction volume =

Polymerease Chain Reactio

The PCRs were performed using

Deep vent was mostly used as b

Thermostability: half-life of 23 ho

template in a 50 l reaction volume

Overlapping PCR-

The overlap extension polymera

specific mutations at specific poi

largerpolynucleotide.

It involves 2 PCRs with 2 template

as a template. PCR1-Using LysG rev

reverse primers primer. Now usi

pBAD18R and pBAD18F.

Fig 7: Di

principle

15

product digested purified-6.5l, T4 DNA

0l

n (PCR)-

ither Taq DNA polymerase or Deep Vent

cause it has got High-Fidelity: 5X greater

rs at 95C. Approximately, 10-20 ng of pl

containing 0.2 mM dNTPs and 1 picomole/

se chain reaction is a variant ofPCR.

ts in a sequence or to splice smaller

specific primers and 2 vectors specific prim

erse & pBAD18 forward primers, PCR 2-Usi

g PCR 1 product as a template and am

grammatic representation showing the gene

of overlapping PCR

ligase enzyme-1l, 10mM

proofreading polymerase.

than Taq, Extremely High

smid DNA was used as a

l of each primer.

It is used to insert

DNA fragments into a

rs using LysG (pHYD2676)

g LysG forward & pBAD18

lify using vector specific

ral


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16

PCR amplification for LysG mutant construction-

PCR 1 PCR2 PCR 1+2

COMPONENTSVOLUME

(l)COMPONENTS

VOLUME

(l)COMPONENTS

VOLUME

(l)

10X Thrmo pol

buffer5

10X Thrmo pol

buffer5

10X Thrmo pol

buffer5

dNTPs 1 dNTPs 1 dNTPs 1

Deep Vent

enzyme0.5

Deep Vent

enzyme0.5

Deep Vent

enzyme0.5

LysGR 1 LysGF 1 pBAD18R 1

pBAD18F 1 pBAD18R 1 pBAD18F 1

Template DNA 10 Template DNA 10 PCR 1 product 21

MB grade water 31.5 MB grade water 31.5 PCR 2 product 20.5

Total reaction

volume50l

Total reaction

volume50l

Total reaction

volume50l


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17

Reaction conditions-

PCR 1 and PCR 2 PCR 1+2

ConditionTemperatures

( C )Time

Temperatures

( C )Time

Initial Denaturation 95 5min 95 5min

Denaturation 95 1min 95 1min

Annealing 50 1min 50 1min

Extension 72 1min 72 1min 30sec

Final extension 72 5min 72 5min

Hoarding 4 5min 4 5min

Number of cycles 29 29

Colony PCR-

We can use this type of PCR to identify which are the colonies those have the insert of interest. First add

reverse and forward primers to MB grade water. Now pick up a little bit of colony with micropipettes and

resuspend in the water-primer mixture. Now lysis of the resuspended mixture has been done by heating

in 99C in PCR machine. After that add 10ul of Dream taq that is a mixture ofDreamTaq DNA Polymerase,

optimized Dream Taq buffer, MgCl2 and dNTPs. After that set up for PCR in a reaction condition

mentioned below then check insert release with the help of gel electrophoresis.


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18

COMPONENTS VOLUME in l

pBAD18F 0.5

pBAD18R 0.5

Template DNA (colonies resuspended

in MB grade water)9

Dream Taq 10

Total reaction volume 20l

Reaction conditions-

PCR purification-

The PCR purification protocol of QIAGENTM

kit was employed.

Steps Temperatures ( C ) Time

Initial Denaturation 94 4 min

Denaturation 94 1min

Annealing 50 45sec

Extension 72 1min 30sec

Final extension 72 5 min

Hoarding 4 5 min

Number of cycles 25


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Agarose gel electrophoresi

The DNA samples were mixed with

lblue, 0.25% xylene cyanol and 30

agarose gel in 1X TAE buffer at 5V/

for 30 min at room temperature a

size markers (GeneRuler) of 1kb a

Ladders-

Fig 8: Images of 1kb

Gel elution-

The gel elution protocol of QIAGEN

19

-

the appropriate volumes of the 6X loading b

% glycerol in water) and subjected to elec

cm for 2-4 hours. The gel was stained in 1

d bands were visualized under UV light. C

nd HindIII lamba DNA were run for visualizat

HindIII-DNA

ladder and HindIII lambda DNA.

M gel extraction kit was employed.

uffer (0.25% bromopheno

trophoresis through 0.8%

g/ml of ethidium bromide

mmercially available DNA

ion of DNA.


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20

GENETIC TECHNIQUES-

Transformation (Calcium chloride method)

For routine plasmid transformation where very high efficiencies were not essential, the following methodwhich is a modification of the procedure described by Cohen et alwas used. An overnight culture of the

recipient strain was sub-cultured in fresh LB broth and grown at 37C till middle logarithmic phase. Cells

were washed with cold 0.1 M CaCl2 and resuspended in the same. The cells were incubated on ice for 30-

45 min. 0.1 ml of this cell suspension was mixed with 0.1-0.5 g of the DNA, incubated further for 30 min

and given a heat shock for 90 seconds at 42C. The cultures were rapidly chilled on ice for 1-2 min, mixed

with 1 ml of LB and incubated at 37C for 45-60 min. The cells were pelleted by centrifugation and

resuspended in 300 l of LB. 0.1 ml of this mixture was plated on selective media.

Transformation with Ultra competent cell-


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21


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Stored DH5-Ultracom cells are made by following protocol and 100l of freezing competent are taken.

Then thaw in ice for sometimes followed by the addition of 10l ligated plasmid, mix it properly and

incubate on ice for 45min. After that heat shock is given at 42C for 90sec. The cultures were rapidly

chilled on ice for 1-2 min, mixed with 1 ml of LB and incubated at 37C for 45min. Now after 45min

samples are spin it down at 6000 for 5min. The cells were pelleted by centrifugation and resuspended in

200l of LB. 0.1 ml of this mixture was plated on selective media and incubate overnight at 37C.

-gal assay-

From the purified plate we have to make primary

culture in minimal media with water, B1, MgSO4, 20%

glucose, Ampicilin, Trimethoprim. After that overnight

grown cultures are spin down at 7000 rpm for 3min.

Then the supernatant is discarded and washed with

minimal media.

Now the strains were sub-cultured in minimal media with water, B1, MgSO4, 20% arabinose, 80% glycerol,

Ampicilin, Trimethoprim. Then 3 sets are prepared one with arginine, one with lysine and one without any

amino acid. Now they are allowed to grow. Whenever the O.D of the subculture is 0.3-0.7 we must take

out the cultures and measure the O.D at 600nm. Then a reaction mixture is prepared with 700l of Z

buffer and 300l of cultures. Now to that mixture add few drops of 0.1% SDS and chloroform followed by

mixing with vortex. Now keep the samples in 28C for 10-15min. Then add 200l ONPG (ortho-

Nitrophenyl--galactoside) to mixture, mixed well and the time is noted. When pale yellow colour is

developed then check the O.D (must be ranging from 0.3-0.9). Now add 500l of Na2CO3 to stop the

reaction. Now measure the O.D at 429nm and calculate with the help of the given formula-

-gal unit=


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X-gal assay-

X-gal (5-bromo-4-chloro-indoly

of galactose linked to a substit

hydrolyzed by the -galactosida

gal, when cleaved by -galacto

The latter then spontaneously d

an intensely blue product whi

colored product therefore may

This easy identification of an ac

to be used as a reporter gene in

23

--D-galactopyranoside) is an organic

ted indole. X-gal is an analog of lactose

se enzyme which cleaves the -glycosi

idase, yields galactose and 5-bromo-4-

imerizes and is oxidized into 5,5'-dibro

h is insoluble. X-gal itself is colorless

be used as a test for the presence of a

tive enzyme allows the gene for -galac

arious applications.

compound consisting

, and therefore may be

ic bond in D-lactose. X-

chloro-3-hydroxyindole.

mo-4,4'-dichloro-indigo,

, the presence of blue-

active -galactosidase.

tosidase (the lacZgene)


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RESULTS

Cloning of lysG gene-

Specific mutation inserted in lysG

extracted PCR products as well a

pBAD18 have restriction enzyme si

and ligated plasmids are transfor

plates. Plasmid was isolated from

digesting with HindIII and EcoRI. All

the digestion was partial, as evide

insert (960bp). Plasmid from two o

PCR 1and PCR 2 results of ov

Fig 9:

(A) Sh

(B) Sh

(A)

24

gene by overlapping PCR and pBAD18 used

the vector plasmids are digested as in t

es for HindIII and EcoRI. Now after digesti

ed in DH5 ultra competent cell and wer

eight transformants to check for insert r

the clones selected showed insert release a

t from the presence of linearised plasmid (

the clones was transformed into MC4100

erlapping PCR-

Gel electrophoresis images of PCR products

owing L274S mutant.

owing T94L mutant.

(B)

as a vector. Now the gel

e LysG clone and vector

on ligation was performed

e selected on LB-amp-glu

elease of size 960 bp by

s shown in Fig.9., however

5.6Kb), vector (4.6Kb) and

rgP/argO-lac strain.


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PCR 1+2 results of overlappi

(A)

Fig 10

(A) Sh

(B) Sh

(C) P1

L274S mutants

25

ng PCR-

(B)

(C)

: Gel electrophoresis images of PCR product

owing L274S mutant.

owing T94L mutant.

08S mutant.

T94L, V

muta

s

44M

ts


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Gel showing digested PCR pr

Fig 11: Gel electrophore

Gel showing insert release a

26

oducts and vector plasmids-

is image of pBAD18 digested and dige

ter digestion of transformed clone

sted PCR products.

mutants-

Fig 12: Gel

electrophoresis image

showing that out of 6

transformed colonies

colony 1 and 3 got the

insert release.


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Fig 13: Gel electrophoresis image s

insert release.

Gel showing insert release a

A Colon PCR of L274S m

(C)Colony PCR of P108S mutant

1 2 3

1 2 3 4 5 6

27

owing that out of 8 transformed colonies co

ter colony PCR of transformed clon

Fig 14: Gel electrophoresis iL274S mutant colony 1, 3,

of 8 transformed colonies. (

mutant colony 1, 3, 4, 6, 9,

of 10 transformed colonies.

mutant colony 2, 3, 4, 5, 6,

out of 10 transformed colon

tantsB Colon PCR of T9

s

4 5 6 1 2 3 4 5

7 8

lony 2 and 4 got the

ed mutants-

mage (A) Showing that inhave insert release out

) Showing that in T94L

10 have insert release out

(C) Showing that in P108S

7, 8 have insert release

ies.

4L mutants

6 7 8 9 10


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28

Se quen cin g res ults of Clo nes -

L274S CLONE-

Fig 14: Sequencing results shownthat that lysine (CTG) has been changed

to serine (CTC).

T94L CLONE-

Fig 15: Sequencing results shown that

Threonine (ACC) has been changed to Leucine

(TTA).


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29

P108S CLONE-

Fig 16: Sequencing results shown that Proline (CCC) has been changed to Serine (TCT).

X-gal assay results of Clones-

All positive regulators (L274S, P108S, T94L) are giving Blue coloration in the X-gal plate but not in same intensity

while negative regulators (V144M) giving white colouration and we used LysG as our control which is giving white

coloration in X-gal plate.

Fig 17: X-gal assay results of clones


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30

- galac to si dase as say re sul t

Table 5: -gal assay of different strains-

Strain OD600 Time (t) OD420 OD420 avg. -gal unit

1. Control 1 (Z

buffer)

0.00

0.00

60min 0.001 0.001 00

2. Control 2 ( Zbuffer)

60min 0.001

3. ArgP (Glu-Arg) 0.720 60min 0.012 0.013 1.00

4. ArgP (Glu-Arg) 60min 0.014

5. ArgP (Glu-Lys) 0.810 60min 0.026 0.028 1.92

6. ArgP (Glu-Lys) 60min 0.030

7. ArgP (Ara-Arg) 0.303 60min 0.018 0.020 3.73

8. ArgP(Ara-Arg) 60min 0.023

9. ArgP (Ara-Lys) 0.473 60min 0.011 0.016 1.88

10. ArgP (Ara-Lys) 60min 0.02111. ArgP +(Glu-Arg) 0.293 90min 0.109 0.115 14.55

12. ArgP +(Glu-Arg) 90min 0.122

13. ArgP +(Glu-Lys) 0.274 90min 0.012 0.15 2.05

14. ArgP +(Glu-Lys) 90min 0.018

15. ArgP +(Ara-Arg) 0.298 90min 0.122 0.128 16.00

16. ArgP+(Ara-Arg) 90min 0.134

17. ArgP +(Ara-Lys) 0.252 90min 0.013 0.016 2.35

18. ArgP+ (Ara-Lys) 90min 0.019

19. pBAD18 (Glu-

Arg) 0.452

60min 0.022

0.021 2.59

20. pBAD18 (Glu-

Arg)

60min 0.020

21. pBAD18 (Glu-Lys) 0.632 60min 0.019 0.205 1.81

22. pBAD18 (Glu-Lys) 60min 0.022

23. pBAD18 (Ara-

Arg)

0.391 60min 0.017

0.015 2.14

24. pBAD18 (Ara-

Arg)

60min 0.014

25. pBAD18 (Ara-Lys) 0.356 60min 0.021 0.024 4.1226. pBAD18 (Ara-Lys) 60min 0.027

27. +pBAD18 (Glu-Arg) 0.483 90min 0.308 0.291 22.31

28. +pBAD18 (Glu-Arg) 90min 0.275

29. +pBAD18 (Glu-Lys) 0.489 90min 0.041 0.037 3.08

30. +pBAD18 (Glu-Lys) 90min 0.033

31. +pBAD18 (Ara-Arg) 0.457 90min 0.308 0.320 26.66

32. +pBAD18 (Ara-Arg) 90min 0.333

33. +pBAD18 (Ara-Lys) 0.409 90min 0.033 0.03 2.77

34. +pBAD18 (Ara-Lys) 90min 0.027


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31

[Strains used in the -galactosidase assay-

1) ArgP = ArgP strain, 2) ArgP+= ArgP

+strain, 3) pBAD18= pBAD18 transformed in ArgP/argO-lac,

4) +pBAD18 = pBAD18 transformed in ArgP+/argO-lac, 5) LysG = LysG transformed in ArgP/argO-lac,

6) L274S = L274S mutant transformed in ArgP/argO-lac, 7) P108S = P108S mutant transformed in

ArgP/argO-lac 8) T94L = T94L mutant transformed in ArgP/argO-lac.]

35. LysG (Glu-Arg) 0.273 90min 0.012 0.03 4.28

36. LysG (Glu-Arg) 90min 0.018

37. LysG (Glu-Lys) 0.296 90min 0.023 0.024 3.03

38. LysG (Glu-Lys) 90min 0.026

39. LysG (Ara-Arg) 0.247 90min 0.030 0.029 4.1440. LysG (Ara-Arg) 90min 0.028

41. LysG (Ara-Lys) 0.209 90min 0.022 0.023 3.15

42. LysG (Ara-Lys) 90min 0.024

43. L274S (Glu-Arg) 0.491 60min 0.407 0.43 51.8

44. L274S (Glu-Arg) 60min 0.453

45. L274S (Glu-Lys) 0.450 60min 0.24 0.26 3.20

46. L274S (Glu-Lys) 60min 0.28

47. L274S (Ara-Arg) 0.462 60min 0.587 0.600 72.28

48. L274S (Ara-Arg) 60min 0.614

49. L274S (Ara-Lys) 0.419 60min 0.020 0.44 5.8650. L274S (Ara-Lys) 60min 0.068

51. P108S (Glu-Arg) 0.714 100min 0.447 0.45 21.42

52. P108S (Glu-Arg) 100min 0.443

53. P108S (Glu-Lys) 0.511 100min 0.029 0.027 1.761

54. P108S (Glu-Lys) 100min 0.025

55. P108S (Ara-Arg) 0.240 100min 0.432 0.459 65.5756. P108S (Ara-Arg) 100min 0.486

57. P108S (Ara-Lys) 0.896 100min 0.044 0.038 1.413

58. P108S (Ara-Lys) 100min 0.032

59. T94L (Glu-Arg) 0.568 80min 0.505 0.518 39.84

60. T94L (Glu-Arg) 80min 0.532

61. T94L (Glu-Lys) 0.664 80min 0.031 0.04 2.66

62. T94L (Glu-Lys) 80min 0.039

63. T94L (Ara-Arg) 0.652 80min 0.529 0.508 32.56

64. T94L (Ara-Arg) 80min 0.487

65. T94L (Ara-Lys) 0.591 80min 0.027 0.03 2.12

66. T94L (Ara-Lys) 80min 0.033


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Graphical representation

Fig 18: Graphical representation o

that LysG mutants regulates the ar

Fig 19: Graphical representation sh

as a control.

0

10

20

30

40

50

60

70

80

-gal

unit

32

f the -gal assay result-

LysG regulation level of argO from -gal a

gO in high level in the presence of arginine

owing the LysG regulation levels of LysG mu

Strains

say. It has been observed

o-factor.

tants using LysG wild type

b-gal unit


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33

DISCUSSION

Specific mutations were inserted in lysG (pHYD2676) gene by overlapping PCR and pBAD18 used as a

vector. Now the gel extracted PCR products as well as the vector plasmids are digested as in the LysG

clone and vector pBAD18 have restriction enzyme sites for HindIII and EcoRI. Now after digestion

ligation was performed and ligated plasmids are transformed in DH5 ultra competent cell and were

selected on LB-amp-glu plates.

As several ArgP-dominant (ArgPd) variants that confer elevated Arg-independent argO expression, ArgP

(S94L), ArgP (P108S) and ArgP (P274S) activates lysEexpression in E. coli. So we constructed several LysG

mutants at the corresponding residues of LysG namely T94L, P108S and L274S to determine if they

regulate argO or not. After inserting the specific mutation the plasmids carrying the mutations are

transformed into ArgP-argO-lac and ArgP+-argO-lac strain and -galactosidase assay was performed.

From the assay result it has been concluded that in the presence of arginine all those mutants activates

the transcription of argO. So we can infer that LysG mutants regulate argO in the presence of arginine

with both arabinose and glucose.


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REFERENCE

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2. Madhusudan R. Nandineni and J. Gowrishankar. 2004 Evidence for an Arginine Exporter

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Escherichia coli. JOURNAL OF BACTERIOLOGY 186.11.35393546.

3. Nandineni, M. R., R. S. Laishram, and J. Gowrishankar. 2004. Osmosensitivity associated with

insertions in argP (iciA) or glnE in glutamate synthase-deficient mutants ofEscherichia coli. J.

Bacteriol. 186:6391-6399.

4. Carmelita N. Marbaniang and J. Gowrishankar. 2011. Role of ArgP (IciA) in Lysine-Mediated

Repression in Escherichia coli. JOURNAL OF BACTERIOLOGY. 191: 59855996.

5. Bellmann, A., M. Vrljic, M. Patek, H. Sahm, R. Kramer, and L. Eggeling. 2001. Expression

control and specificity of the basic amino acid exporter LysE ofCorynebacterium glutamicum.

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transcription of the Escherichia coli dapB gene by preventing its activation by the ArgP

activator. J. Bacteriol. 190:5224-5229.

7. Celis, R. T. 1999. Repression and activation of arginine transport genes in Escherichia coliK 12

by the ArgP protein. J. Mol. Biol. 294:1087-1095.

8. Goss, T. J. 2008. The ArgP protein stimulates the Klebsiella pneumoniae gdhA promoter in a

lysine-sensitive manner. J. Bacteriol. 190:4351-4359.

9. Momany, C., and E. L. Neidle. 2012. Defying stereotypes: the elusive search for a universal

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