The Effect of The Effect of IPTG Levels IPTG Levels on Expression on Expression of the Lac of the Lac OperonOperon

Joel BradshawJoel Bradshaw

W.F. West High SchoolW.F. West High School

Gene Regulation: The Basics

• Controls the production of proteinsControls the production of proteins

• Can be controlled by the presence of Can be controlled by the presence of certain chemicals, or can be self-certain chemicals, or can be self-regulatingregulating

• Examples: ROP, Lac OperonExamples: ROP, Lac Operon

Lac Operon: The Problem

• E. coliE. coli prefers prefers GlucoseGlucose

• Other energy sources availableOther energy sources available

• Must be digestedMust be digested

• Energy wasteEnergy waste

HHHHHHHHHHHHHHHH

Lac Operon: The Solution

• ββ-Galactosidase-Galactosidase (Z) breaks down (Z) breaks down lactoselactose

• More More ββ-Galactosidase-Galactosidase needed needed

• Only when Only when GlucoseGlucose is not available is not available

HHHHHHHHHHHHHHHH

The Lac Operon – How it works

• Three main regionsThree main regions– LacILacI site produces the site produces the repressorrepressor– CAPCAP site produces the site produces the Catabolite Activator Catabolite Activator

ProteinProtein– LacZYALacZYA region produces the protein and region produces the protein and

contains binding sitescontains binding sites

HHHHHHHHHHHHHHHH

HHHHHHHHHHHHHHHH

Negative Control

• LacI RegionLacI Region produces produces repressorrepressor

• RepressorRepressor binds to binds to operatoroperator

• When When polymerasepolymerase attaches to attaches to promoterpromoter, , repressorrepressor stops transcription stops transcription

HHHHHHHHHHHHHHHH

Negative Control

• ββ-Galactosidase -Galactosidase digests digests lactoselactose

• Break down of Break down of lactoselactose causes causes allolactoseallolactose

• AllolactoseAllolactose releases releases repressorrepressor

• More lactose broken downMore lactose broken down

NOT SO MUCH DETAIL!LacZ is replaced with GFP! – move to questions

Positive Control

• Cyclic AMPCyclic AMP binds to binds to CAPCAP– Compound helps bind Compound helps bind polymerasepolymerase to to promoterpromoter

• GlucoseGlucose inhibits formation of inhibits formation of cyclic AMPcyclic AMP

• Polymerase achieves little transcriptionPolymerase achieves little transcription

HHHHHHHHHHHHHHHH

NO HIGH LEVELS TWICE!

Positive Control

• GlucoseGlucose levels are low - levels are low - cyclic AMP cyclic AMP can can formform

• Cyclic AMPCyclic AMP binds to the binds to the CAPCAP– Helps Helps polymerase polymerase bind to bind to promoterpromoter

• PolymerasePolymerase can transcribe can transcribe

HHHHHHHHHHHHHHHH

How it applies

• IPTGIPTG imitates imitates allolactoseallolactose

• GFPGFP inserted into LacZYA inserted into LacZYA

HHHHHHHHHHHHHHHH

ROP: The Problem

ROP: The solution

• The ROP region controls the copying of The ROP region controls the copying of the plasmidthe plasmid

• Ensures a low copy numberEnsures a low copy number

• Eliminates the variable number of Eliminates the variable number of plasmids in a bacteria, ensuring regular plasmids in a bacteria, ensuring regular measurementsmeasurements

• The ROP protein, increases the affinity of The ROP protein, increases the affinity of the primers for each other, so they don’t the primers for each other, so they don’t stick to the Origin of Replication, stick to the Origin of Replication, causing the plasmid to cease replicating.causing the plasmid to cease replicating.

ROP

OR

I

ROP: How it works

ROPO

RI

Hypothesis

Increased IPTG Increased IPTG concentration will result concentration will result in increased expression in increased expression

of the Lac Operonof the Lac Operon

Questions

• Effect of IPTG concentration on lac expressionEffect of IPTG concentration on lac expression– Does IPTG affect lac expression?Does IPTG affect lac expression?– How does it affect expression?How does it affect expression?– Is there a limit to how much IPTG affects?Is there a limit to how much IPTG affects?

Experimental Design

• Engineer a plasmid with the ROP and the Engineer a plasmid with the ROP and the Lac OperonLac Operon

• Grow the plasmid in varying Grow the plasmid in varying concentrations of IPTGconcentrations of IPTG

• Measure the amount of GFP productionMeasure the amount of GFP production

Creating the pNGNp

UC

19

pB

R3

22

GF

P

Creating the pNGN

• Amplified desired sectionsAmplified desired sections

• Restricted pBR & pUCRestricted pBR & pUC

GF

Pp

BR

pU

C

2612 bp

23130

9416

6557

4361

2322

2027

564

707 bp

• Ligated pBR and pUCLigated pBR and pUC• Transformed ligations into Transformed ligations into E. coliE. coli• Spread on plates with ampicillin to Spread on plates with ampicillin to

select for bacteria with pUC19select for bacteria with pUC19

Creating the pNGN

Mar

ker

10 u

l30

ul

pB

R R

est.

pU

C R

est.

Creating the pNGN

• Plucked colonies from Plucked colonies from plates and grew in OSC’splates and grew in OSC’s

• Extracted, restricted to Extracted, restricted to separate targeted sectionsseparate targeted sections

• #5 has pUC and pBR #5 has pUC and pBR fragments, and nothing elsefragments, and nothing else

Ma

rke

r#

1#

2#

3#

4#

5M

ark

er

23130

9416

6557

4361

2322

2027

564

2612 bp 707 bp

Creating the pNGN

• Ligated #5 and GFP fragmentsLigated #5 and GFP fragments• Transformed ligations into Transformed ligations into E. coliE. coli• Spread on platesSpread on plates

– Ampicillin (pUC)Ampicillin (pUC)– IPTG (GFP)IPTG (GFP)

• Selected 10 glowing coloniesSelected 10 glowing colonies• Grew in OSC’sGrew in OSC’s

Creating the pNGN

• Extracted DNA from Extracted DNA from OSC’s and restricted to OSC’s and restricted to separate fragmentsseparate fragments

• Selected #6 due to Selected #6 due to concentrationconcentration

100

bp

Mar

ker

#1

#2

#3

#4

#5

M

arke

r

#6

#7

#8

#9

#10

100

bp

Mar

ker

Creating the pNGN

• Sent pNGN to be Sent pNGN to be sequencedsequenced

• Both ROP & GFP Both ROP & GFP verifiedverified

Experimental Design

• Experimental groupsExperimental groups– Negative ControlNegative Control

• E. coliE. coli with no plasmid with no plasmid

• E. coliE. coli with no plasmid + 10µl IPTG with no plasmid + 10µl IPTG

– IPTG levels (µl per 5 ml broth)IPTG levels (µl per 5 ml broth)• 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 200, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20

• Grew conicals for several hoursGrew conicals for several hours

Measuring Fluorescence

• Pelleted cellsPelleted cells

• Resuspended cellsResuspended cells

• Measured in Measured in fluorometerfluorometer

Results

• As IPTG increased, fluorescenceAs IPTG increased, fluorescenceincreased – to a pointincreased – to a point

• Once IPTG reached a threshold, fluorescence dropped off and slowly decreasedOnce IPTG reached a threshold, fluorescence dropped off and slowly decreased

-40

-20

0

20

40

60

80

100

0 2 4 6 8 10 12 14 16 18 20

A

A (2)

B

B (Adj)

Average

Poly. (Average)

Results

• Fluctuations, but still peaked near 10Fluctuations, but still peaked near 10• Increase after 18Increase after 18

0 2 4 6 8 10 12 14 16 18 20+ IP T G

-50

-25

0

25

50

75

100

125

150

175

200

T ria l 2

M eas. 1

M eas. 2

Experim ental G roup

RF

U

Results

The Effect of IPTG Concentration over Time

10

100

1000

2:00 2:30 3:00 3:30 4:15 5:05 5:45 15:21

0

10

20

30

DH5a

Statistical Analysis

Fluorescence Time 0 10 20 30 E. coli Correlation

2:00 23 19 17 18 28 -0.834

2:30 28 37 40 28 24 0.063

3:00 22 38 40 49 28 0.954

3:30 26 45 51 63 34 0.979

4:15 29 56 60 59 40 0.822

5:05 33 98 122 102 41 0.772

5:45 55 188 182 173 46 0.710

15:21 256 540 710 1296 137 .968

  59.000 127.625 152.750 223.500 47.250 0.986

P-Value 0.242 0.129 0.093 0.084

Discussion

• Increased IPTG = increased Increased IPTG = increased GFP GFP (LacZYA)(LacZYA) production production

• Expression reduced at high levelsExpression reduced at high levels– Loss of function due to wasted energyLoss of function due to wasted energy– Reduced GFP productionReduced GFP production

Limitations

• Trials 1 & 2Trials 1 & 2– Amount of time grownAmount of time grown

• Trial 3Trial 3– Amount of initial brothAmount of initial broth– Length of measurement periodLength of measurement period

Future Research

• Compare IPTG to LactoseCompare IPTG to Lactose

• Test effects of glucoseTest effects of glucose

• Test farther out to investigate the bump Test farther out to investigate the bump in second trialin second trial

• Control time grownControl time grown

Acknowledgements

• Mr. Henri Weeks, W.F. West High SchoolMr. Henri Weeks, W.F. West High School

• My Family, for tolerating my long My Family, for tolerating my long absencesabsences

• The FedEx lady, for waiting to take our The FedEx lady, for waiting to take our samples for sequencingsamples for sequencing

Questions?

pUC19

pBR322

pGLO

Misc

• CorrelationCorrelation– Upward: 0.942507Upward: 0.942507– Downward: 0.87545Downward: 0.87545– Curve: RCurve: R22 = .8478 = .8478

• IPTG: IPTG: IsopropylthiogalactoIsopropylthiogalactopyranosidepyranoside

HHHHHHHHHHHHHHHH

Purpose of the Lac Operon

• The genes in ZYA digest The genes in ZYA digest lactoselactose– Z = Z = ββ-Galactosidase-Galactosidase (breaks down (breaks down lactoselactose))

• GlucoseGlucose• GalactoseGalactose

– Y = Y = Lactose PermeaseLactose Permease (transports lactose into cell) (transports lactose into cell)– A = Thiogalactoside transacetylase (toxicity)A = Thiogalactoside transacetylase (toxicity)

• A side effect of the break down of A side effect of the break down of lactoselactose is is allolactoseallolactose

• AllolactoseAllolactose binds to binds to repressorrepressor and relelases itand relelases it

NOT SO MUCH DETAIL!LacZ is replaced with GFP! – move to questions

Results

  0 10 20 30 E. coli 0 10 20 30 E. coli

8:00 23 19 17 18 28 0.074 0.072 0.073 0.069 0.082

8:30 28 37 40 28 24 0.072 0.081 0.086 0.078 0.074

9:00 22 38 40 49 28 0.124 0.131 0.125 0.121 0.121

9:30 26 45 51 63 34 0.151 0.163 0.166 0.154 0.146

10:15 29 56 60 59 40 0.217 0.220 0.235 0.214 0.199

11:05 33 98 122 102 41 0.303 0.322 0.318 0.304 0.314

11:45 55 188 182 173 46 0.423 0.435 0.436 0.423 0.410

21:21 256 540 710 1296 137 0.882 0.772 0.805 0.866 0.970

  0.94039 0.96432 0.95419 0.93601 0.97552          

Fluorescence E. Coli density