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

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

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...

• 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 with brand new colonies and to store these new glycerol stocks at -80°C.

We prepared 5 ml cultures (re-filling the remaining 250 ul of the grown bacteria) 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. Colony PCR for P0440(3 colonies) and R0040(3 colonies) plates to check for contaminations. Gel run for the resulting reactions.

Susanna preparing samples for colony PCR

Colony PCR: P0040 insert was amplified correctly, while R0040 showed contaminants or unexpected bands

• Gel results: the 3 colonies of P0440 all have the plasmid with the correct length of the insert (1078 bp), while there have been problems with R0040 colonies. R0040-1 and R0040-2 show the correct length of the insert (292 bp) with a high weight contaminant and R0040-3 show an unexpected wrong length. Next week we are going to digest P0440 and R0040 to perform their assembly, so we will check the actual plasmid and insert length again.

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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.

Red pellet for J23100-seq and normal-coloured pellet for B0030 after 7 min at 7000 rpm centrifuge

3OC6-HSL 2 mM vial

Experiment with Tecan F200

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

June, 6th

Experiment with Tecan F200

• Results check