IGEM:IMPERIAL/2006/LabCalendar/2006-8-31

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(Chemostat)
 
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Make up test media dilutions for the chemostat. I will make a dilution series of diferent LB concentrations (keeping salts the same) to get a lower OD, idealy around 0.3 / 0.4 '''Then tomorrow''' I will use glycerol to increace the nutriant concentration of the media so the LB becomes carbon limiting, trying to get a glycerol conc of 0.8. '''Today '''I will also make media (LB + Glycerol that can support a population up to an OD of >2.8 '''[[User:JohnChattaway|JohnChattaway]] 04:25, 31 August 2006 (EDT)'''
Make up test media dilutions for the chemostat. I will make a dilution series of diferent LB concentrations (keeping salts the same) to get a lower OD, idealy around 0.3 / 0.4 '''Then tomorrow''' I will use glycerol to increace the nutriant concentration of the media so the LB becomes carbon limiting, trying to get a glycerol conc of 0.8. '''Today '''I will also make media (LB + Glycerol that can support a population up to an OD of >2.8 '''[[User:JohnChattaway|JohnChattaway]] 04:25, 31 August 2006 (EDT)'''
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Resluts in Testing section.
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Results can be found [[IGEM:Imperial/Results/Growth_Media|here]].
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Current revision



TO DO:
*Testing of T9002, J37016, J37020, S01656, J37015, J37015RS
*Culture new ligated part J37024 for mini & maxiprep on Friday
*Miniprep 'old' J37024 (both plates)



Contents

FIRST TIME: Biosensor Testing

  • Following the Biosensor protocol, the acylase dilutions were set up - they are stored in the freezer
  • The AHL/enzyme mix was set up
  • Dr. O'Hare offered us to use the biosensor in his lab, so took over the samples on ice
  • Take pH samples off ice & measure pH
Sample Name pH readings
after 16min
pH readings
after 20min
pH readings
after 31min
Control (no enzyme added)
MiliQ 8.0 7.9 7.9 7.9
G 8.2 7.8 7.7 7.8
K 7.9 7.9 . 7.9
. 7.4 7.4 . 7.6
O 7.9 7.9 . 7.9
. 7.7 8.2 . 8.2
  • PROBLEM: Since the pH electrode is too large, the volume of 1mL is not enough to submerge the sensor fully in the analyte
  • Thus, the above readings are not reliable
  • Experiment has to be repeated with larger volumes (at least 2mL), and the electrode does not fit into an eppendorf


  • Afterwards, the enzyme activity was assessed by using the relevant protocol (similar to T9002)
  • Placed in the shaker at 17:00 - to be taken out at 21:00
  • Froze cells overnight, to read GFP expression tomorrow

Chemostat

Make up test media dilutions for the chemostat. I will make a dilution series of diferent LB concentrations (keeping salts the same) to get a lower OD, idealy around 0.3 / 0.4 Then tomorrow I will use glycerol to increace the nutriant concentration of the media so the LB becomes carbon limiting, trying to get a glycerol conc of 0.8. Today I will also make media (LB + Glycerol that can support a population up to an OD of >2.8 JohnChattaway 04:25, 31 August 2006 (EDT) Results can be found here.

Testing T9002/J37016/J37015

  • OD of o/n T9002 culture: 2.00
  • OD of o/n J37016 culture: 2.00
  • OD of o/n J37020 culture: 2.00

Put in shaker at 9:40. To be taken out at 11:40.

After 2hrs

  • OD of T9002: 0.827 (innoculate 3.023ml culture into 16ml)
  • OD of J37016: 0.947 (innoculate 2.640ml culture into 16ml)
  • OD of J37020: 0.806 (innoculate 3.102ml culture into 16ml)

Put in the shaker at 12:10. To be taken out at 4:10.

Miniprep

  • 4 J37024 1 samples
  • 4 J37024 2 samples
  • 3 J37018 samples

J37024 ligations appear to be unsuccessful. Currently being religated (J37024 from scratch-PCR stage).

Culturing

  • Culture up the repeated ligation of j37024 (II) to maxi and mini tomorrow.


To do tomorrow

  • Maxiprep J37024 II
  • Ligate to form J37025
  • Re ligate to form part J37018

BL21 competent cells

  • In order to determine whether the reason for lack of protein expression is due to the faulty aiiA gene or whether it is due to the DH5α cells not being able to express the protein succesfully, we have decided to transform the part <BBpart>S01656</BBpart> into BL21 competent cells.
  • We transformed it using heat shock, as these cells respond better to this method of transformation compared to electroporation.
  • They will need to be cultured up tomorrow (01/09) and then we able to do a Western blot on Monday.
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