Team:Newcastle/Notebook/Lab/Lightbulb/Week 14

September 19th 2016

We inoculated fresh liquid LB broth for use with the plate reader and testing the battery constructs:

  • Battery 1
  • Battery 2
  • Lightbulb construct BBa_K1895000 (PhtpG regulated lightbulb)
  • Alternative light construct BBa_K1895006 ((PdnaK regulated lightbulb)
  • Variable Resistor (tagged)
  • Variable Resistor (un-tagged)
  • Positive Control BBa_KI20270 (from InterLab study)
  • Interlab study Sample 1 (from InterLab study)
  • Negative Control (from InterLab study)
  • BL21 cells

September 20th 2016

We started out by setting up a plate using the same layout as previously described in order to test our ‘light bulb’ constructs, using the fresh cultures we inoculated the day before. We used a 96 well plate with clear bottoms and black sides in order to measure both OD600 and fluorescence. We diluted the concentrated cell mixture to an appropriate OD600 value of around 0.05, which will allow us to see a full growth curve. We used a plate reader with the gain set to 528 (as used in the inter-lab study), measuring fluorescence at two wavelengths, an excitation of 485nm for both and emissions of 518nm and 538nm respectively. Over the next few days we plan to test the constructs in the plate reader at 30°C, 37°C and 42°C over a 24-hour period. Today we tested at 37°C. Inoculated fresh LB culture for use on the following day.

September 21st 2016

Today we looked at the data collected over night, the RFU of some of many of the wells had reached a maximum intensity that the plate reader was able to detect after around 4 hours. This was due to the gain value being too high as these tests were carried out on a different plate reader to that used in the inter-lab study. Inoculated fresh LB culture for use on the following day.

September 22nd 2016

We again used the plate reader using the same protocol as described above but this time with the gain value set at 250. Inoculated fresh LB culture for use on the following day.

September 23rd 2016

Again many of the wells had saturated the detection limits of the plate reader. This time when we set up the plate reader for overnight we used an auto gain adjustment feature on the system. Inoculated fresh LB culture for use on the following day.