The Effect of IPTG Levels on Expression of the Lac Operon by 48232d97

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									The Effect of
IPTG Levels
on Expression
of the Lac
Operon
Joel Bradshaw
W.F. West High School
Gene Regulation: The Basics

• Controls the production of proteins
• Can be controlled by the presence of
  certain chemicals, or can be self-
  regulating
• Examples: ROP, Lac Operon
Lac Operon: The Problem

•   E. coli prefers Glucose
•   Other energy sources available
•   Must be digested
•   Energy waste
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Lac Operon: The Solution

• β-Galactosidase (Z) breaks down lactose
• More β-Galactosidase needed
• Only when Glucose is not available
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The Lac Operon – How it works

• Three main regions
  – LacI site produces the repressor
  – CAP site produces the Catabolite Activator
    Protein
  – LacZYA region produces the protein and
    contains binding sites
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Negative Control

• LacI Region produces repressor
• Repressor binds to operator
• When polymerase attaches to promoter,
  repressor stops transcription
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Negative Control

•   β-Galactosidase digests lactose
•   Break down of lactose causes allolactose
•   Allolactose releases repressor
•   More lactose broken down
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Positive Control

• Cyclic AMP binds to CAP
  – Compound helps bind polymerase to promoter
• Glucose inhibits formation of cyclic AMP
• Polymerase achieves little transcription
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Positive Control

• Glucose levels are low - cyclic AMP can
  form
• Cyclic AMP binds to the CAP
  – Helps polymerase bind to promoter
• Polymerase can transcribe
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How it applies

• IPTG imitates allolactose
• GFP inserted into LacZYA
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ROP: The Problem
ROP: The solution

• The ROP region controls the copying of
  the plasmid
• Ensures a low copy number
• Eliminates the variable number of
  plasmids in a bacteria, ensuring regular
  measurements
ROP: How it works

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




                                  ORI
                            ORI
       Hypothesis

     Increased IPTG
concentration will result
in increased expression
    of the Lac Operon
Questions

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

• Engineer a plasmid with the ROP and the
  Lac Operon
• Grow the plasmid in varying
  concentrations of IPTG
• Measure the amount of GFP production
Creating the pNGN




                          pBR322


                                   pUC19
                    GFP
Creating the pNGN

• Amplified desired sections
• Restricted pBR & pUC




                                 pBR
                                 pUC
                                 GFP
                         23130
                         9416
                         6557
                         4361
                         2322
                         2027
                          564




    2612 bp     707 bp
Creating the pNGN




                                        pBR Rest.
                                        pUC Rest.
• Ligated pBR and pUC




                                        Marker
                                        10 ul
                                        30 ul
• Transformed ligations into E. coli
• Spread on plates with ampicillin to
  select for bacteria with pUC19
Creating the pNGN




                                Marker




                                Marker
• Plucked colonies from




                                #3
                                #1
                                #2

                                #4
                                #5
  plates and grew in OSC’s               23130


• Extracted, restricted to
                                         9416
                                         6557


  separate targeted sections             4361
                                         2322


• #5 has pUC and pBR
                                         2027
                                         564


  fragments, and nothing else




      2612 bp       707 bp
Creating the pNGN
• Ligated #5 and GFP fragments
• Transformed ligations into E. coli
• Spread on plates
  – Ampicillin (pUC)
  – IPTG (GFP)
• Selected 10 glowing colonies
• Grew in OSC’s
Creating the pNGN

• Extracted DNA from




                            100 bp Marker
                            100 bp Marker




                                  Marker
  OSC’s and restricted to




                               #10
                               #1
                               #2
                               #3
                               #4
                               #5

                               #6
                               #7
                               #8
                               #9
  separate fragments
• Selected #6 due to
  concentration
Creating the pNGN

• Sent pNGN to be
  sequenced
• Both ROP & GFP
  verified
Experimental Design
• Experimental groups
   – Negative Control
      • E. coli with no plasmid
      • E. coli with no plasmid + 10µl IPTG
   – IPTG levels (µl per 5 ml broth)
      • 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20
• Grew conicals for several hours
Measuring Fluorescence

• Pelleted cells
• Resuspended cells
• Measured in
  fluorometer
Results
 100


                                               A
                                               A (2)
 80
                                               B
                                               B (Adj)
                                               Average
                                               Poly. (Average)
 60




 40




 20




  0
       0   2   4   6   8   10   12   14   16           18        20



 -20




 -40



• As IPTG increased, fluorescence
  increased – to a point
• Once IPTG reached a threshold, fluorescence
  dropped off and slowly decreased
      Results
                                       Trial 2
      200
      175
      150
      125
      100
RFU




      75
                                                                           Meas. 1
      50                                                                   Meas. 2
      25
       0
      -25
      -50
            0   2   4   6   8     10    12   14      16   18   20
                                                                    +
                                                                    IPTG
                                Experimental Group

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

           The Effect of IPTG Concentration over Time
1000




                                                               0
                                                               10
 100                                                           20
                                                               30
                                                               DH5a




  10
    2:00     2:30   3:00   3:30   4:15   5:05   5:45   15:21
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 GFP
  (LacZYA) production

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

• Trials 1 & 2
  – Amount of time grown


• Trial 3
  – Amount of initial broth
  – Length of measurement period
Future Research

• Compare IPTG to Lactose
• Test effects of glucose
• Test farther out to investigate the bump
  in second trial
• Control time grown
Acknowledgements

• Mr. Henri Weeks, W.F. West High School
• My Family, for tolerating my long
  absences
• The FedEx lady, for waiting to take our
  samples for sequencing
Questions?
pUC19
pBR322
pGLO
Misc

• Correlation
  – Upward: 0.942507
  – Downward: 0.87545
  – Curve: R2 = .8478
• IPTG:
  Isopropylthiogalacto
  pyranoside
Purpose of the Lac Operon
• The genes in ZYA digest lactose
  – Z = β-Galactosidase (breaks down lactose)
     • Glucose     NOT SO MUCH DETAIL!
     • Galactose   LacZ is replaced with GFP! –
                   move to questions
  – Y = Lactose Permease (transports lactose into cell)
  – A = Thiogalactoside transacetylase (toxicity)
• A side effect of the break down of lactose is
  allolactose
• Allolactose binds to repressor
  and relelases it
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  Results
                            Fluorescence                                          E. Coli density
           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

								
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