IGEM:MIT/2005/Input: Ligand

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==POC==
==POC==
 +
http://www.ci.berkeley.ca.us/environmentalhealth/images/bacteria.gif
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<br>
Maxine
Maxine
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==Device Parts==
==Device Parts==
-
* A pair of ssDNA strands complementary to each other, each bound to a fluorescein molecule at the 5' end, of the following lengths:
 
<table>
<table>
-
<tr><td>
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<tr>
 +
<td>
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Lengths:<p>
10 bp<br>
10 bp<br>
12 bp<br>
12 bp<br>
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[[Image:DNA.gif]]
[[Image:DNA.gif]]
</td></tr><table>
</td></tr><table>
 +
* A pair of ssDNA strands complementary to each other, each bound to a fluorescein molecule at the 5' end, of various lengths.
 +
<p>
 +
http://www.biocompare.com/images/techart/Qiagen_Endofree_fig1.jpg
==Current Status==
==Current Status==
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:* <b> Troubling Data </b>
:* <b> Troubling Data </b>
::cell membrane premeability ~ 600 Daltons ~ 6 amino acids [http://www.rzuser.uni-heidelberg.de/~u53/abstracts/macdonald.html Source]
::cell membrane premeability ~ 600 Daltons ~ 6 amino acids [http://www.rzuser.uni-heidelberg.de/~u53/abstracts/macdonald.html Source]
-
::DNA base pair average molecular weight ~ 650 Dalton [http://www.growtall.com/technical-data3.htm and http://www.eppendorf.com  Source]
+
::DNA base pair average molecular weight ~ 650 Dalton [http://www.growtall.com/technical-data3.htm Source] and [http://www.eppendorf.com  Source]
::Fluorescein: 330 daltons
::Fluorescein: 330 daltons
::Also, see "DNA specs" under "Maxine's Notes" Section
::Also, see "DNA specs" under "Maxine's Notes" Section
Line 61: Line 67:
:* <b> Possible Solutions</b>
:* <b> Possible Solutions</b>
-
::* <b>Is ligand is linear enough (i.e. it has a small cross-sectional area) that it can still fit through the outer membrane despite its large mass? </b>
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::*<b>Is ligand linear enough (i.e. it has a small cross-sectional area) to fit through the outer membrane despite its large mass? </b>
:::Fluorescein dimers for Epo receptor: best linker ~ 45 A
:::Fluorescein dimers for Epo receptor: best linker ~ 45 A
:::DNA diameter: ~26 A
:::DNA diameter: ~26 A
-
:::"Antisense agents are 10 or more bp, and this length is typically too large for efficient passive cellular uptake by diffusion across lipid bilayers" http://www.jbc.org/cgi/content/full/277/9/7144
+
:::"Antisense agents are 10 or more bp, and this length is typically too large for efficient passive cellular uptake by diffusion across lipid bilayers" [http://www.jbc.org/cgi/content/full/277/9/7144 Source]
 +
::*<b>Use Bacillus Subtilis instead of E. Coli</b>
 +
:::Permeability estimated to be 25kDa.  "The Permeability of the Wall Fabric of E. Coli and Bacillus subtilis"
 +
:::Use DNA uptake system??
 +
::::[http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=372826&tools=bot Genetic competence]
 +
::::[http://textbookofbacteriology.net The Bacillus Genus]
::*<b>Use other types of inputs</b>
::*<b>Use other types of inputs</b>
-
:::NPN as possible replacement for fluorescein? See [[../NPN/]]
+
:::NPN as possible replacement for fluorescein? [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=89666 Paper] [[../NPN/]]
-
:::Nitrocefin as poss replacemeht for fluorescein? See [[../Nitrocefin/]]
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:::Nitrocefin as poss replacemeht for fluorescein? [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=89666 Paper] [[../Nitrocefin/]]
:::scFV that binds to isoketal adducts [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15082070 Paper ]  [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=56783236 Sequence]
:::scFV that binds to isoketal adducts [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15082070 Paper ]  [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=56783236 Sequence]
:::<s>Digoxin</s>: 780 Da -- too heavy
:::<s>Digoxin</s>: 780 Da -- too heavy
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:::*EDTA treatment
:::*EDTA treatment
:::*Heatshock
:::*Heatshock
 +
 +
::*<b>DNA transporter: bacterial conjugation--too complicated??</b>
==Experiments==
==Experiments==
-
[[../Input reception Experiments/]]
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[[../Some initial fluorescene uptake results/]]<br>
-
*Move information in link to respective place.
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[[../Old Preliminary Dimer Uptake Experiment Protocol/]]
 +
 
 +
===Preliminary Experiment: Uptake of Fluorescein and Fluorescein Dimers by Chemically Competent and Non-competent Bacteria===
 +
<b>Materials</b>
 +
*Competent cells (taken from Tom Knight's Lab)
 +
*Plate of MC4100 cells
 +
*M9 media
 +
*Fluorescein solution
 +
*Fluorescein dimer solution
 +
*Microscope: cover slips, cover slides, wells, agarose gel, penny
 +
*Shaker
 +
 
 +
<b>Procedure</b>
 +
#Grow cells overnight in m9 media from the MC4100 plate.
 +
#Make up the following solutions: 5 micromolar solution of fluorescein dimers in m9, 5 micromolar solution of fluorescein in m9
 +
#Take OD of overnight culture and competent cells.  (If using the nanodrop machine multiply the reading by 10 to obtain the OD.)  Take the correct ml of culture to obtain an eventual 20 microliter sample with a concentration of ~6 X 10^8 cells/ml.
 +
##6 X 10^8 cells/ml*(20 microliters)*(1ml/1000 microliters)*( OD here )= ________ ml of culture
 +
#Take the amount calculated above of overnight and competent cell culture and pipet into 6 labelled eppendorf tubes, 3 overnight, 3 competent.  Centrifuge 1 min, 13,000 rpm.  Pour off supernatant.
 +
#Resuspend competent cells and overnight cells in 5 micromolar fluorescein solution, 5 micromolar fluorescein dimer solution, and M9, making a total of 6 tubes.
 +
#Incubate on bench for 1 hour on shaker.
 +
##While waiting, make agarose gel pads.  To make these, place a cover slip on top of a penny, then drop a small amount of agarose onto the cover slip. Place another cover slip on top, and then wait for agarose gel to harden.
 +
#Centrifuge 1 min, 13,000 rpm and resuspend cells in m9 media.
 +
#Microscope: Peel off the top cover slip of the agarose gel pad.  Cut a small piece of the agarose using a razor, drop a 5 microliter sample onto the small piece of gel, then flip upside down and place in well. 
 +
#Data collection
 +
##Take a bright field and fluorescent picture for each sample, making a total of 12 pictures
 +
#Image processing: for each picture...
 +
##Make sure to keep an iplab version saved
 +
##Select a region of the background and note average fluorescence level
 +
##Select 2 fluorescent cells and note average fluorescence level
 +
 
 +
===Fluorescein Dimer Uptake and Cell Viability Experiment===
 +
<b> Materials </b>
 +
*MC4100 cells
 +
*EDTA solution
 +
*Solution of competent MC4100 cells
 +
*Electroporator
 +
*LB media
 +
*M9 media
 +
*Fluorescein dimer solution (dimer + LB) (concentrations based on results of Will's experiment (see below)
 +
*Fluorescein solution (Fluorescein + LB) (")
 +
 
 +
<b>Procedure </b>
 +
#Culture MC4100 cells overnight in LB
 +
#Centrifuge cells, remove supernatant, resuspend in M9 media
 +
#Dilute cells (concentrations??)
 +
#Split into 5 aliquots.  Label 9 tubes and plates
 +
#Control 1: Centrifuge and resuspend aliquot 1 in fluorescein dimer solution.  Note volume and concentration.
 +
#Control 2: Centrifuge and resuspend aliquot 2 in fluorescein solution. 
 +
#heatshock: Put aliquot 3 in 42 degrees C shaking water bath for 1 minute.  Centrifuge and resuspend in fluorescein dimer solution.
 +
#CaCl2: Take competent cells (of the same volume and concentration as step 5) and centrifuge and resuspend in fluorescein dimer solution.
 +
#EDTA: Put aliquot 4 in 0.8 mM EDTA.  Centrifuge and resuspend cells in fluorescein dimer solution.
 +
#Electroporation: Put aliquot 5 in electroporator (?)
 +
#Cacl2 + heatshock: Put competent cells (same volume and concentration as step 5) into 42 degrees C shaking water bath for 2 min.  Centrifuge and resuspend in fluorescein dimer solution.
 +
#Cacl2 + EDTA: Put competent cells (same volume and concentration as step 5) into 0.8 mM EDTA.  Centrifuge and resuspend cells in fluorescein dimer solution.
 +
#Cacl2 + EDTA + heatshock: Do step 11, and then place in shaking water bath, 42 degrees C, 2 min.  Centrifuge and resuspend cells in fluorescein dimer solution.
 +
#Wait for cells to recover (place on roller drum at 37 degrees for 15 minutes).
 +
#Make appropriate dilution for plating, then plate a portion of contents of each of the 9 tubes.
 +
#Make slides of contents of 9 dubes and check for fluorescence.
 +
 
 +
===Input Reception===
 +
<b>Questions</b>
 +
# Is our fluorescein dimer entering into the cell?
 +
# Distance in space between flurs. -- might depend on 3D conformation/wobblyness
 +
# Separate out single from double stranded
 +
 
 +
===Conclusion===
 +
Mixed results for data.  Not clear if competent cells can take up input more readily than regular cells can.
 +
[http://www.fisheroligos.com/tec_flc.htm Fluorescein information]
==Open Issues==
==Open Issues==
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==Maxine's Notes==
==Maxine's Notes==
-
===(this section is purely for myself so that I have a spot to put my own work)===
+
(this section is purely for myself so that I have a spot to put my own work)
-
 
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[[../Maxine's Notes/]]
-
<b> Key Facts </b>
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-
#Figure out if/how the ligand can pass through the outer membrane of the E.coli cell
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-
*the permeability of the cell membrane is 600 Daltons, which is about 6 amino acids
+
-
::http://www.rzuser.uni-heidelberg.de/~u53/abstracts/macdonald.html
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-
*Fluorescein dimers were designed with linker lengths: 8 mer (27.2 angstroms) to 14 mer (47.6 angstroms)
+
-
::In mammalian paper under "must read" on the main page
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-
*Best linker: 12/13 mer (though all induced growth)
+
-
:: In mammalian paper under "must read" on the main page
+
-
*The average molecular weight of DNA base pair is 650 Dalton
+
-
:: http://www.growtall.com/technical-data3.htm and http://www.eppendorf.com
+
-
*Fluorescein: 330 daltons
+
-
*DNA specs: [[Image:DNA.jpg|700px]]
+
-
 
+
-
<b> Other Background Info </b>
+
-
*[http://www.arches.uga.edu/~dlg/gm-ve.gif Picture] belongs to University of Georgia.
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-
**Outer membrane: This lipid bilayer is found in Gram negative bacteria and is the source of lipopolysaccharide (LPS). LPS is toxic and turns on the immune system;  LPS is found in Gram negative, but not in Gram positive, bacteria.
+
-
**Cell wall is composed of peptidoglycan (polysaccharides + protein), the cell wall maintains the overall shape of a bacterial cell. The three primary shapes in bacteria are coccus (spherical), bacillus (rod-shaped) and spirillum (spiral). Mycoplasma are bacteria that have no cell wall and therefore have no definite shape.
+
-
**Periplasm: This cellular compartment is found only in those bacteria that have both an outer membrane and plasma membrane (e.g. Gram negative bacteria). In the space are enzymes and other proteins that help digest and move nutrients into the cell.
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**Inner membrane: also known as plasma membrane. This is a lipid bilayer much like the cytoplasmic (plasma) membrane of other cells. There are numerous proteins moving within or upon this layer that are primarily responsible for transport of ions, nutrients and waste across the membrane.
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*Problems
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**The outer membrane is the major permeability barrier in Gram negative bacteria. Gram negative bacteria store '''degradative enzymes''' in the periplasmic space.
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*Solutions
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**Work with E.Coli strain that is cell-wall deficient
+
-
**Or work with E.Coli strain that has the most permeable cell wall <-- LIMITATION of antigen size. Ways to make cell wall permeable are: treatment with chemicals (example: EDTA)
+
-
**[[../Linkers/]]
+
-
*Statistics:
+
-
**Cell wall: in Bacillus licheniformis and Bacillus subtilis, a diffusional radius of not more than 2.5 nm for molecules is determined ([http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=126549&query_hl=4 Estimates of the porosity of Bacillus licheniformis and Bacillus subtilis cell walls])
+
-
**Cell outer membrane: in E.Coli and Salmonella Typhimurium, molecules of 600 Da and less diffuse through.
+
-
*[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=14763988&query_hl=2 Peptidoglycan hydrolytic activities associated with bacteriophage virions.] paper with some in depth characterization of ways around/through the periplasm. maybe we can learn something from how phages shoot through the periplasm to do their bizness. (unread)
+

Current revision

Contents

POC

bacteria.gif
Maxine

Function

  • To design an input for receiver unit 1 (ToxR) and receiver unit 2 (FecA)
    • Receiver 1:
      • To design a input (ligand) with 2 fluorescein molecules attached by a piece of DNA, which will be used in the intermediate step of testing if binding of an antigen to our system can cause dimerization and subsequent transcription of the desired output gene.
    • Receiver 2:
      • Input here is simply just a fluorecein molecule to test if binding of an antigen to our system can cause a conformation change and subsequent transcription of the desired output gene.

Device Depiction

fluorescein.png


Fluorescein Structure

Device Parts

Lengths:

10 bp
12 bp
15 bp
20 bp </td>

Image:DNA.gif

</td></tr>
  • A pair of ssDNA strands complementary to each other, each bound to a fluorescein molecule at the 5' end, of various lengths.

Qiagen_Endofree_fig1.jpg

Current Status

Completed Work

  1. Determined the length and content of DNA between fluorescein molecules
    1. 10 bp ~ 33.2 A: because DNA twists every 33.2 A, at this point, the fluorescein molecules, on different strands of DNA, will be pointing directly away from each other, 180 degrees apart
    2. 12 bp ~ 39.2 A: fluorescein molecules will be pointing 90 degrees apart
    3. 15 bp ~ 50.2 A: fluorescein molecules will point in the same direction
    4. 20 bp ~ 66.4 A: fluorescein molecules will point 180 degrees away from each other
  2. Obtained sequence of puc19 and used it for DNA construct
  3. Ordered from Invitrogen, with TK's help--should arrive Friday, Jul 15

Steps to take

  1. Figure out if/how the ligand can pass through the outer membrane of the E.coli cell
    1. Make cell wall more permeable with electroporation, heatshock cells, or find cells with mutation in the outermembrane??
    2. Find other types of inputs that are small enough to fit into the cell
  2. Determine if RE sites should be added into the piece of DNA (for negative testing of dimerization)

Current Work

  • Figure out if/how the ligand can pass through the outer membrane of the E.coli cell
  • Troubling Data
cell membrane premeability ~ 600 Daltons ~ 6 amino acids Source
DNA base pair average molecular weight ~ 650 Dalton Source and Source
Fluorescein: 330 daltons
Also, see "DNA specs" under "Maxine's Notes" Section
  • Possible Solutions
  • Is ligand linear enough (i.e. it has a small cross-sectional area) to fit through the outer membrane despite its large mass?
Fluorescein dimers for Epo receptor: best linker ~ 45 A
DNA diameter: ~26 A
"Antisense agents are 10 or more bp, and this length is typically too large for efficient passive cellular uptake by diffusion across lipid bilayers" Source
  • Use Bacillus Subtilis instead of E. Coli
Permeability estimated to be 25kDa. "The Permeability of the Wall Fabric of E. Coli and Bacillus subtilis"
Use DNA uptake system??
Genetic competence
The Bacillus Genus
  • Use other types of inputs
NPN as possible replacement for fluorescein? Paper NPN
Nitrocefin as poss replacemeht for fluorescein? Paper Nitrocefin
scFV that binds to isoketal adducts Paper Sequence
Digoxin: 780 Da -- too heavy
  • Increase cell wall permeability:
  • Mutation in outer membrane: "Lipoprotein Mutation Accelerates Substrate Permeability-Limited Touluene Dioxygenase-Catalyzed Reaction," Biotechnology Progress (article not available online--see Maxine for copy of printed version)
  • Attachment of cell-permeabilizing peptide to our input Paper PNA Paper Review
  • Electroporation
  • EDTA treatment
  • Heatshock
  • DNA transporter: bacterial conjugation--too complicated??

Experiments

Some initial fluorescene uptake results
Old Preliminary Dimer Uptake Experiment Protocol

Preliminary Experiment: Uptake of Fluorescein and Fluorescein Dimers by Chemically Competent and Non-competent Bacteria

Materials

  • Competent cells (taken from Tom Knight's Lab)
  • Plate of MC4100 cells
  • M9 media
  • Fluorescein solution
  • Fluorescein dimer solution
  • Microscope: cover slips, cover slides, wells, agarose gel, penny
  • Shaker

Procedure

  1. Grow cells overnight in m9 media from the MC4100 plate.
  2. Make up the following solutions: 5 micromolar solution of fluorescein dimers in m9, 5 micromolar solution of fluorescein in m9
  3. Take OD of overnight culture and competent cells. (If using the nanodrop machine multiply the reading by 10 to obtain the OD.) Take the correct ml of culture to obtain an eventual 20 microliter sample with a concentration of ~6 X 10^8 cells/ml.
    1. 6 X 10^8 cells/ml*(20 microliters)*(1ml/1000 microliters)*( OD here )= ________ ml of culture
  4. Take the amount calculated above of overnight and competent cell culture and pipet into 6 labelled eppendorf tubes, 3 overnight, 3 competent. Centrifuge 1 min, 13,000 rpm. Pour off supernatant.
  5. Resuspend competent cells and overnight cells in 5 micromolar fluorescein solution, 5 micromolar fluorescein dimer solution, and M9, making a total of 6 tubes.
  6. Incubate on bench for 1 hour on shaker.
    1. While waiting, make agarose gel pads. To make these, place a cover slip on top of a penny, then drop a small amount of agarose onto the cover slip. Place another cover slip on top, and then wait for agarose gel to harden.
  7. Centrifuge 1 min, 13,000 rpm and resuspend cells in m9 media.
  8. Microscope: Peel off the top cover slip of the agarose gel pad. Cut a small piece of the agarose using a razor, drop a 5 microliter sample onto the small piece of gel, then flip upside down and place in well.
  9. Data collection
    1. Take a bright field and fluorescent picture for each sample, making a total of 12 pictures
  10. Image processing: for each picture...
    1. Make sure to keep an iplab version saved
    2. Select a region of the background and note average fluorescence level
    3. Select 2 fluorescent cells and note average fluorescence level

Fluorescein Dimer Uptake and Cell Viability Experiment

Materials

  • MC4100 cells
  • EDTA solution
  • Solution of competent MC4100 cells
  • Electroporator
  • LB media
  • M9 media
  • Fluorescein dimer solution (dimer + LB) (concentrations based on results of Will's experiment (see below)
  • Fluorescein solution (Fluorescein + LB) (")

Procedure

  1. Culture MC4100 cells overnight in LB
  2. Centrifuge cells, remove supernatant, resuspend in M9 media
  3. Dilute cells (concentrations??)
  4. Split into 5 aliquots. Label 9 tubes and plates
  5. Control 1: Centrifuge and resuspend aliquot 1 in fluorescein dimer solution. Note volume and concentration.
  6. Control 2: Centrifuge and resuspend aliquot 2 in fluorescein solution.
  7. heatshock: Put aliquot 3 in 42 degrees C shaking water bath for 1 minute. Centrifuge and resuspend in fluorescein dimer solution.
  8. CaCl2: Take competent cells (of the same volume and concentration as step 5) and centrifuge and resuspend in fluorescein dimer solution.
  9. EDTA: Put aliquot 4 in 0.8 mM EDTA. Centrifuge and resuspend cells in fluorescein dimer solution.
  10. Electroporation: Put aliquot 5 in electroporator (?)
  11. Cacl2 + heatshock: Put competent cells (same volume and concentration as step 5) into 42 degrees C shaking water bath for 2 min. Centrifuge and resuspend in fluorescein dimer solution.
  12. Cacl2 + EDTA: Put competent cells (same volume and concentration as step 5) into 0.8 mM EDTA. Centrifuge and resuspend cells in fluorescein dimer solution.
  13. Cacl2 + EDTA + heatshock: Do step 11, and then place in shaking water bath, 42 degrees C, 2 min. Centrifuge and resuspend cells in fluorescein dimer solution.
  14. Wait for cells to recover (place on roller drum at 37 degrees for 15 minutes).
  15. Make appropriate dilution for plating, then plate a portion of contents of each of the 9 tubes.
  16. Make slides of contents of 9 dubes and check for fluorescence.

Input Reception

Questions

  1. Is our fluorescein dimer entering into the cell?
  2. Distance in space between flurs. -- might depend on 3D conformation/wobblyness
  3. Separate out single from double stranded

Conclusion

Mixed results for data. Not clear if competent cells can take up input more readily than regular cells can. Fluorescein information

Open Issues

  • Is fluorescein too big to get into the periplasm?
  • Important to note that the optimal distance of 12/13 mer was for the EpoR protein.. ToxR might have a different optimal distance.

Need Help With

Issue: can we ignore the fact that our construct is too many Daltons by assuming that according to the geometry, our construct is linear enough to pass through the outer membrane?

  • Find people with experience w/cell membrane permeability of e. coli
  • Find different way to link fluorescein molecules

Maxine's Notes

(this section is purely for myself so that I have a spot to put my own work)

Maxine's Notes

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