Week from May 25th, to May 31st, 2009

May, 25th

• iGEM starts now!!!

• We planned to dedicate the first two weeks to prepare a glycerol stock for every BioBrick of interest for our project. The following weeks will be dedicated to the BioBrick assembly.

• DNA resuspension from iGEM 2009 plates. We resuspended all the high quality constitutive promoter family members from UCBerkeley collection and GFP protein generator:

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

May, 26th

• All the 15 incubated plates showed colonies!

Moreover, many UCBerkeley promoter family members showed red fluorescent colonies, according to the ranking of promoters' estimated strength (see http://partsregistry.org/Promoters/Catalog/Anderson).

High quality Berkeley promoters and E0240 plates: high yield for all of them!

J23102 plate: the promoter is very strong and RFP expression could be seen in the red-coloured colonies. The same result was seen in the other strong Berkeley promoters

• We picked one colony from each plate and infected 1 ml of LB + Amp. We incubated the inocula at 37°C, 220 rpm for 5 and 1/2 hours.

• After 5 hours and 1/2 all the cultures were turbid.

• Glycerol stocks for the cultures, that were in their exponential growth phase (750 ul of bacteria and 250 ul of 80% glycerol).

Preparation of the first experiment with Tecan F200

• We re-filled with 5 ml of LB + Amp the remaining 250 ul of the grown bacterial cultures containing:
  • J23100, J23101, J23118 (strong constitutive promoters with RFP protein generator downstream) to test fluorescence detection of the microplate reader;
  • E0240 (negative control because bacteria bearing this BioBrick don't express fluorescent proteins)

May, 27th

• We wanted to resuspend Q04400 and Q04121 in order to build up respectively an aTc and an IPTG/lactose sensor, but we realized that MIT QC classified their sequences as "inconsistent"...We searched for smaller BioBricks to re-build Q04400 and Q04121 with a minimum number of assemblies:
  • Q04400 = P0440 (available in iGEM 2009 plates) + R0040 (available in iGEM 2009 plates)
  • Q04121 = P0412 (not available in iGEM 2009 plates) + R0011 (available in iGEM 2009 plates)

• DNA resuspension from iGEM 2009 plates. We resuspended three lysis-involved parts, a double transcriptional terminator and lacZ transcriptional unit in order to build up a beta-galactosidase protein generator (last year, a beta-galactosidase protein generator was available in 2008 Distributon - I732950 - but its sequence was almost completely wrong):

• Only B0015 and I732017 QC sequence analysis was available...we hoped that the three lysis parts' sequences were correct!

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

Experiment with Tecan F200

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

May, 28th

May, 29th

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