Week from June 1st, to June 7th, 2009

June, 3rd

• We received lactose monohydrate, M9 minimal salts and Thiamine Hydrochloride from Sigma.

• DNA resuspension from iGEM 2009 plates. We resuspended the two parts we needed to re-built the inconsistent BioBrick Q04400, the five commonly used RBSs and a 3OC6-HSL inducible measurement system for GFP test at Tecan F200:

B0030 B0031 P0440 (tetR protein generator) B0032 B0033 T9002 (AHL-inducible GFP) B0034 R0040 (Ptet)

Matteo and Lorenzo resuspending BioBricks from iGEM 2009 plates

• Transformation of resuspended DNA (2 ul) in TOP10 E. coli, plated transformed bacteria and incubated the plates overnight at 37°C.

• We streaked a plate with a 2008 glycerol stock containing E0240 under the control of a constitutive promoter (we call this construct "01") in order to use it in the fluorescence test at Tecan F200 on June 5th.

June, 4th

• All the overnight plates showed colonies. Unfortunately, B0034 and B0032 plates also showed a very small amount of unexpected red colonies...(@_@!?) These two parts had been resuspended from iGEM 2009 plate 1, just like Berkeley constitutive promoters, but it is not clear how they could be contaminated...

• 01 plate showed green fluorescence under UV rays, as we expected because GFP was expressed constitutively!

• Anyway, we decided to continue our work and we picked one colony from the following plates:

and infected 1 ml of LB + Amp. We incubated the inocula at 37°C, 220 rpm for 5 and 1/2 hours. Of course, we carefully avoided to pick red colonies from B0034 and B0032 plates:) Sequencing checks will tell us if our parts are correct.

• Glycerol stocks for all the grown cultures (except for 01, because we already had it).

• NOTE: we re-picked colonies from J23100, J23101 and J23118 plates because data analysis of experiment 1 at Tecan F200 showed that J23101 promoter seemed stronger than J23100. This is not in accordance to the ranking of the promoters (http://partsregistry.org/Promoters/Catalog/Anderson), so we decided to repeat the test at Tecan F200 and to store these now glycerol stocks at -80°C.

• We prepared 5 ml cultures for:
  • B0030-seq (for sequencing because it is the first RBS we wanted to use)
  • J23100-seq (for sequencing because we wanted to check the integrity of the downstream RFP)
  • J23100 (for tomorrow's test - RFP assay)
  • J23101 (for tomorrow's test - RFP assay)
  • J23118 (for tomorrow's test - RFP assay)
  • E0240 (for tomorrow's test - negative control)
  • T9002 (NOT INDUCED for tomorrow's test - GFP assay to detect leaky activity caused by Plux - we will call it T9002-)
  • T9002 (TO INDUCE TOMORROW for the test - GFP assay - we will call it T9002+)
  • 01 (for tomorrow's test - positive control)

• and incubated them overnight at 37°C, 220 rpm.

June, 5th

• All the overnight cultures were grown. RFP-expressing cultures were red-coloured, as usual!

• Miniprep for B0030-seq and J23100-seq.

• We sent B0030 and J23100 to BMR Genomics for sequencing.

Preparation of the second experiment with Tecan F200

• We diluted 1:10 these overnight cultures:
  • J23100
  • J23101
  • J23118
  • E0240
  • T9002-
  • T9002+
  • 01

• We induced the diluted and non-diluted cultures of T9002+ with 3OC6-HSL 200 nM.

• We incubated all the diluted and non-diluted cultures at 37°C, 220 rpm for 4 and 1/2 hours.

Experiment with Tecan F200

• Description
• Purpose:
• Materials & Methods
• Protocol
• Results

June, 6th

Experiment with Tecan F200

• Results check