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, picked from the plate stored at +4°C) 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.

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

• Download Protocol

June, 6th

Experiment with Tecan F200

• Results check