Difference between revisions of "Team:Queens Canada/Protocols"

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  <h2>Materials</h2>
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<div style="background-color:#afbee8;color:white;padding:50px 150px 50px 150px;">
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  <ul>
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<center>
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  <li>Guanidinium chloride (GdmCl)</li>
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<h1 style="color:black;"><b> Relating Relative Promoter Units to Transcription Rate </b></h1><br><hr><br>
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  <li>47mm polycarbonate filter membranes with 10um pores</li>
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<p align="left"><font size="5" color="black" style="Corbel">  
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  <li>Nuclease solution (Benzonase)</li>
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<em><u> Assumption: </em></u><br>
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  <li>5% (m/v) SDS</li>
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<p align="left"><font size="5" color="black" style="Corbel">  
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  <li>Distilled water</li>
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The first major assumption, is declaring <em>"Promoter Activity" </em>, to be a direct model of transcription rate. "Promoter Activity" is the number of RNAP molecules that clear the final base pair of a promoter (with units of [PoPS]
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  </ul><br>
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= Polymerase per second).</p>  
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<p align="left"><br>Thus,<br>
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<img src="https://static.igem.org/mediawiki/2017/b/b9/T--Queens_Canada--RPUEquation1.png" style = "width:40%"><br>
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Where, <br>
 +
<img src="https://static.igem.org/mediawiki/2017/d/db/T--Queens_Canada--PoPSEquation2.png" style = "width:20%"><br>
 +
Thus, through simple substitution: <br>
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<img src="https://static.igem.org/mediawiki/2017/4/48/T--Queens_Canada--RPUEquationallvariables.png" style = "width:60%"><br>
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<br><br><br></font></p></center>
  
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<p align="left" style="line-height:1.5;"><font size="5" style="corbel" color="black">
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  <li>After the induction period, add guanidinium chloride to the induction culture to reach a final concentration of 0.8M</li>
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The modelling can further be simplified through a series of assumptions: <br>
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  <li>Incubate for 1-2 hours at 4°C prior to filtration</li>
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1) GFP expressed from test and standard promoters, have equivalent maturation rates, as they mature under the same conditions: <br>
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  <li>Withdraw 30-50mL of the Gdm-containing culture, then vacuum filter using 47mm polycarbonate filter membranes with 10um pores</li>
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<img src="https://static.igem.org/mediawiki/2017/a/ae/T--Queens_Canada--alphaassumptionEq.png" style="width:13%">
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  <li>Incubate the filtered biomass with 5mL of 8M GdmCl for 5 minutes</li>
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<br><br>
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  <li>Vacuum filter, then rinse 3 times with DI water</li>
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2) Since both the test and standard promoters are carried on the same plasmid backbone, assume they have the same average copy number: <br>
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  <li>Add 5mL of an aqueous solution (2uM MgCl2) of nuclease to the filtered biomass</li>
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<img src="https://static.igem.org/mediawiki/2017/f/f5/T--Queens_Canada--avgCopyNumberEq.png" style="width:13%"><br> <br>
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  <li>Vacuum filter to remove the liquid then rinse 3 times with 5mL of DI water</li>
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3) Since promoters have been standardized to have identical transcription initiation sites (predicted) and identical sequences downstream of the site, we expect them to produce the same mRNA sequences.<br>
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  <li>Incubate the biomass on the filter with 5mL of 5% (m/v) SDS in water for 5 minutes</li>
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Therefore,<br>
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  <li>Vacuum filter, then rinse 5 times with 5mL DI water</li>
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Expect transcribed mRNA to be identical, implying mRNA degradation rates are equivalent: <br>
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  <li>Scrape the semi-purified curli from the filter paper with a flat spatula</li>
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<img src="https://static.igem.org/mediawiki/2017/d/d0/T--Queens_Canada--mRNADegRateEq.png" style="width:13%"> <br><br>
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  <li>Lyophilize, and store at 4°C</li>
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Thus, we assume translational rates of immature GFP from some mRNA are equal: <br>
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  </ol>
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<img src="https://static.igem.org/mediawiki/2017/8/8a/T--Queens_Canada--proteinDegRateEq.png" style="width:13%"><br><br>
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4) Assume that immature GFP is stable. Therefore, protein degradation is negligable compared to dilution due to cell growth. <br>  
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Thus: <br>  
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<img src="https://static.igem.org/mediawiki/2017/7/74/T--Queens_Canada--dilutionRateEq.png" style="width:13%"><br>
 +
 
 +
Thus, <br>
 +
<img src="https://static.igem.org/mediawiki/2017/2/2b/T--Queens_Canada--RPUEquation2.png" style="width:40%"><br>
 +
and if<br> <img src="https://static.igem.org/mediawiki/2017/b/b3/T--Queens_Canada--lessthanalphaEq.png" style="width:13%"><br>
 +
Then, <br>
 +
<img src="https://static.igem.org/mediawiki/2017/7/73/T--Queens_Canada--alphaapproximation.png" style="width:10%"><br>
 +
<br>
 +
Therefore, we assume the difference between growth rates of cells containing the test promoter construct and cells containing the standard promoter construct, is negligible compared to the maturation rate of GFP. <br><br><hr><br>
 +
Thus, for the purposes of QGEM's modelling; <br>
 +
<img src="https://static.igem.org/mediawiki/2017/a/ac/T--Queens_Canada--RPUFinal.png" style="width:40%">
 +
</font></p>
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<br>
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</div>
 
</div></div>
 
</div></div>
  

Revision as of 23:24, 16 October 2017

Protocols from key experiments for our project:


Relating Relative Promoter Units to Transcription Rate



Assumption:

The first major assumption, is declaring "Promoter Activity" , to be a direct model of transcription rate. "Promoter Activity" is the number of RNAP molecules that clear the final base pair of a promoter (with units of [PoPS] = Polymerase per second).


Thus,

Where,

Thus, through simple substitution:




The modelling can further be simplified through a series of assumptions:
1) GFP expressed from test and standard promoters, have equivalent maturation rates, as they mature under the same conditions:


2) Since both the test and standard promoters are carried on the same plasmid backbone, assume they have the same average copy number:


3) Since promoters have been standardized to have identical transcription initiation sites (predicted) and identical sequences downstream of the site, we expect them to produce the same mRNA sequences.
Therefore,
Expect transcribed mRNA to be identical, implying mRNA degradation rates are equivalent:


Thus, we assume translational rates of immature GFP from some mRNA are equal:


4) Assume that immature GFP is stable. Therefore, protein degradation is negligable compared to dilution due to cell growth.
Thus:

Thus,

and if

Then,


Therefore, we assume the difference between growth rates of cells containing the test promoter construct and cells containing the standard promoter construct, is negligible compared to the maturation rate of GFP.


Thus, for the purposes of QGEM's modelling;


Materials

  • LB media
  • Ice bucket and ice
  • Temperature controlled centrifuge
  • 2 polypropylene JA 10 500mL bottles
  • 15% glycerol
  • 0.6 mL tubes
  • P1000 pipette and tips
  • Stripettes and automatic pipette for larger volumes
  • Shaker at 37 °C, 200rpm

  1. Inoculate 250mL of LB medium with PQN4.
  2. Incubate at 37°C, 200 rpm shaking overnight.
  3. Add 1mL of overnight culture to an 1L flask of LB media and incubate with shaking at 200rpm, 37C.
  4. Incubate until OD600 of around 0.4-0.7 is reached
  5. Before continuing, set up the necessary materials:
    1. Pre-cool centrifuge to 4°C
    2. Prepare ice bucket
    3. Chill 1L MQ water and 15% glycerol buffer on ice
  6. Once the cultures reach desired OD600, take them out of incubation and put them on ice for 30 minutes (the tube should feel cool).
  7. From here on, keep cells always cool, at or below 4°C
  8. After cooling, spin the tubes in a refrigerated (4°C) centrifuge for 30min at 5000rpm.
  9. Pour off supernatant carefully, taking care not to pour off the pellet. If the pellet is not attached to the wall after centrifugation, smear the pellet onto the wall of the tube and centrifuge again using longer centrifugation times. Re-suspend bacteria in 500mL MQ water
  10. Centrifuge again for 30 minutes at 5000 rpm and 4 °C temp
  11. Pour off supernatant again, re-suspend pellet in 250mL MQ water on ice as before.
  12. Centrifuge again for 30 minutes at 5000 rpm, 4 °C.
  13. Pour off supernatant and re-suspend pellet in 10mL ice cold 15% glycerol solution.
  14. Spin in 15mL tubes in large centrifuge for 15 minutes at 3400rpm, 4 °C
  15. Resuspend pellets in 2mL of 15% glycerol mixture
  16. Pipette 50ul aliquots into tubes. Label tubes properly.
  17. Store samples on ice for immediate use or freeze 50ul aliquots in-80°C. According to some reports, the efficiency of electrocompetent cells reduces after each freezing, so immediate use may result in highest efficiencies.

Materials

  • Congo Red powder
  • Brilliant Blue G250 dye
  • Yeast extract powder
  • Casamino acids
  • Agar
  • IPTG
  • Antibiotic of choice

  1. CR stock: dissolve 1 g of Congo Red in 100 mL of water and filter sterilize. Store at 4°C.
  2. Brilliant Blue stock: dissolve 1 g Brilliant Blue G250 dye in 100 mL water and filter sterilize. Store at 4°C. [brilliant blue increases the colour contrast of the colonies on the agar]
  3. YESCA CR agar plates: 10 g/L casamino acids, 1 g/L yeast extract, and 20 g/L agar, 100 ug/mL antibiotic (for Amp; will vary depending on your antibiotic), 0.5 mM IPTG, 50 µg/mL Congo Red and 1 µg/mL Brilliant Blue. Autoclave only the dissolved agar and yeast extract, and filter sterilize the other components. Add the filtered components after autoclaved mixture has cooled.
  4. Pick single colonies and streak out on a YESCA CR agar plate
  5. To induce curli production, grow bacteria on YESCA CR agar at 26°C for 48 h
  6. Check the color of the bacterial colonies. Wild-type curli-producing E. coli cells stain red on YESCA CR agar, whereas curli defective mutants are usually pink or white. E. coli mutants with hyper curli production sometimes stain dark red

Materials

  • Congo Red powder
  • Brilliant Blue G250 dye
  • Yeast extract powder
  • Casamino acids
  • Agar
  • IPTG
  • Antibiotic of choice

  1. CR stock: dissolve 1 g of Congo Red in 100 mL of water and filter sterilize. Store at 4°C.
  2. Brilliant Blue stock: dissolve 1 g Brilliant Blue G250 dye in 100 mL water and filter sterilize. Store at 4°C. [brilliant blue increases the colour contrast of the colonies on the agar]
  3. YESCA CR agar plates: 10 g/L casamino acids, 1 g/L yeast extract, and 20 g/L agar, 100 ug/mL antibiotic (for Amp; will vary depending on your antibiotic), 0.5 mM IPTG, 50 µg/mL Congo Red and 1 µg/mL Brilliant Blue. Autoclave only the dissolved agar and yeast extract, and filter sterilize the other components. Add the filtered components after autoclaved mixture has cooled.
  4. Pick single colonies and streak out on a YESCA CR agar plate
  5. To induce curli production, grow bacteria on YESCA CR agar at 26°C for 48 h
  6. Check the color of the bacterial colonies. Wild-type curli-producing E. coli cells stain red on YESCA CR agar, whereas curli defective mutants are usually pink or white. E. coli mutants with hyper curli production sometimes stain dark red

Materials

  • Congo Red powder
  • Brilliant Blue G250 dye
  • Yeast extract powder
  • Casamino acids
  • Agar
  • IPTG
  • Antibiotic of choice

  1. CR stock: dissolve 1 g of Congo Red in 100 mL of water and filter sterilize. Store at 4°C.
  2. Brilliant Blue stock: dissolve 1 g Brilliant Blue G250 dye in 100 mL water and filter sterilize. Store at 4°C. [brilliant blue increases the colour contrast of the colonies on the agar]
  3. YESCA CR agar plates: 10 g/L casamino acids, 1 g/L yeast extract, and 20 g/L agar, 100 ug/mL antibiotic (for Amp; will vary depending on your antibiotic), 0.5 mM IPTG, 50 µg/mL Congo Red and 1 µg/mL Brilliant Blue. Autoclave only the dissolved agar and yeast extract, and filter sterilize the other components. Add the filtered components after autoclaved mixture has cooled.
  4. Pick single colonies and streak out on a YESCA CR agar plate
  5. To induce curli production, grow bacteria on YESCA CR agar at 26°C for 48 h
  6. Check the color of the bacterial colonies. Wild-type curli-producing E. coli cells stain red on YESCA CR agar, whereas curli defective mutants are usually pink or white. E. coli mutants with hyper curli production sometimes stain dark red