Imperial College London/Notebook/16 September 2009

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(Difference between revisions)
(New page: {{Imperial/09/TemplateTop}} ==Testing== ===Diauxic growth 10th Sep=== ====Protocol considerations==== *Multiwell plate reader **Using script IGEM Abs, taking readings every 30 min *Ov...)
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==Testing==
==Testing==
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===Diauxic growth 10th Sep===
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===Lac characterisation 16th Sep===
====Protocol considerations====
====Protocol considerations====
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*Multiwell plate reader
 
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**Using script IGEM Abs, taking readings every 30 min
 
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*Overnight culture cells were grown. (normal Top10)
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*Overnight cells were diluted in fresh media 1:12
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*A volume of overnight cells were diluted into fresh M9 media
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*The cells were allowed to grow until their OD reached around 0.6 (around 4 hours)
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*IPTG was added from a stock solution of 1M IPTG and  0.1M IPTG.  Note that IPTG takes some time to defrost
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*180ul of Media with relevant IPTG conc was added to each 96 well plate.  20ul of cells were added.
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*Put in the plate reader overnight using protocol IGEM Fluor Abs (do not use)
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*1ml of each secondary carbon source media (in M9) was prepared
 
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*200ul was added to 96 well plate as blank
 
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*Write out well plate layout and do test runs of script *IMPORTANT*
 
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*After about 5 hours, when cells were at a high enough OD (cloudy), cells were added 1:20 into the media
 
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**the cells and media were mixed well, and then 200ul was added into 96 well plates
 
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*The plate reader was run overnight
 
====Results====
====Results====
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[[Image:Diauxic growth2.jpg]]<br>
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[[Image:Lac-RFP.jpg]]<br>
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[[Media:Diauxic growth_10-09.xls| Download raw data]]
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[[Media:Lac-RFP 1609.xls| Download raw data]]
====Conclusions====
====Conclusions====
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*Cells in Maltose grow slower, while the growth rate in the other secondary carbon sources are similar
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*There was no change in the cell growth rate with IPTG
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*The cells appear to have continuous growth up till 7 hours.  However, after that, the results become random.  This is probably due to the evaporation problem
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**Since a Lac-RFP construct was used, this shows that IPTG, for both its toxicity and protein production stress, does not have much effect on cell growth at the concentrations tested
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====Suggestions/Improvements====
====Suggestions/Improvements====
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*There is an initial spike.  This should be a multiplate reader problem
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*The script merged the fluorescence readings into the absorbance readings.  <br>
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**Suggestion is to use a higher starting OD ~ 0.5 or 1
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After overnight readings, the regular absorbance readings were obtained, but no fluorescence readings were obtained.
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Therefore, the results had to be converted to a normal cell growth analysis.
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*The diauxic growth is using normal cells which grow on Strep.  However, the growth profile might be significantly different on Amp (for our testing construct)
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**Try to give the script test runs before leaving them overnight, and check on results regularly if possibly
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**Therefore, use a cell with an Amp construct that is not expressed
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*Try not to use any readings from the multiplate reader after around 8 hours
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*To arrive at the switch point faster, 0.01% Glucose will be used instead for next experiment
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{{Imperial/09/TemplateBottom}}
{{Imperial/09/TemplateBottom}}

Revision as of 22:00, 4 October 2009

Contents

Testing

Lac characterisation 16th Sep

Protocol considerations

  • Overnight cells were diluted in fresh media 1:12
  • The cells were allowed to grow until their OD reached around 0.6 (around 4 hours)
  • IPTG was added from a stock solution of 1M IPTG and 0.1M IPTG. Note that IPTG takes some time to defrost
  • 180ul of Media with relevant IPTG conc was added to each 96 well plate. 20ul of cells were added.
  • Put in the plate reader overnight using protocol IGEM Fluor Abs (do not use)


Results

Lac-RFP.jpg
Download raw data

Conclusions

  • There was no change in the cell growth rate with IPTG
    • Since a Lac-RFP construct was used, this shows that IPTG, for both its toxicity and protein production stress, does not have much effect on cell growth at the concentrations tested


Suggestions/Improvements

  • The script merged the fluorescence readings into the absorbance readings.

After overnight readings, the regular absorbance readings were obtained, but no fluorescence readings were obtained. Therefore, the results had to be converted to a normal cell growth analysis.

    • Try to give the script test runs before leaving them overnight, and check on results regularly if possibly


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