Week from August 3rd, to August 9th, 2009

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August, 3rd

• This week we planned to continue the assembly of the synthetic ethanol producing operon.

• We received DH5alpha, XL1-Blue and DB3.1 competent cells from Bologna iGEM Team. Thank you!!

• We performed 2 screenings for the 7 samples of B3 and for the 7 samples of B4:

• SCREENING 1 - Ojective: check the presence of non-ligated plasmids. We planned not to digest with standard enzymes because the resulting fragments would result either too small or too big. Insert excision would generate a possible 129pb fragment, while vector linearization would generate possible 3Kb or 5Kb fragments. We used ApE to decide the best enzymes to cut. Digestion with XhoI-PstI (6 ul of DNA, final volume 20ul). There are two XhoI restriction sites on pSB1AK3 plasmid: after the cut, we expected two fragments of the same size (~1.1Kbp) and one fragment of ~3Kbp for B3 positive clones (in negative clones the heaviest band would be ~1.4Kbp), while we expected the same two fragments of ~1.1Kbp and a heavier fragment of ~2.5Kbp for B4 positive clones (in negative clones the heaviest band would be ~1.4Kbp).

B3 and B4 digested samples (XhoI-PstI).

• Gel results:
  • Only B3-1 and B3-5 showed the bands with expected length for the correct plasmid.
  • All B4 samples showed the bands with expected lengths!

• SCREENING 2 - Ojective: check the presence of the right insert length for B3 samples, because there was the possibility that B1 vector (pSB1A2) ligated in B0015 vector (pSB1AK3) as an insert. If so, no XbaI site is present in the final plasmid, while correct ligations should show a ~1900bp fragment as an insert. Digestion with XbaI-PstI (2 ul of DNA, final volume 20ul). We decided to perform the reaction for all B3 samples, even if only two were good.

B3 digesed XbaI-PstI.

• Gel results: B3-1 and B3-5 showed the expected bands for pSB1AK3 (~3200 bp) and B1-B0015 ligated insert (~1900bp).

• We sent the following purified DNA samples to BMR Genomics for sequencing:
  • B3-1
  • B3-5
  • B4-2
  • B4-4
• We planned not to perform ligations this week and to wait for BMR Genomics sequencing results, in order to be sure of what we were assembling.

• LB agar plates + Kan preparation.

Preparation of experiment with Tecan F200

• We inoculated 10 ul of A1, A2, A7, J23100, J23101 and J23118 glycerol stocks in 5 ml of LB + Amp, while we used a single colony from B0030 native plate to infect 5 ml of LB + Amp.

• We incubated these inocula overnight (37°C, 220 rpm).

August, 4th

Preparation of experiment with Tecan F200

• We diluted 1:1000 the overnight cultures of A1, A2, A7, J23100, J23101, J23118 and B0030.

• We incubated the diluted cultures for 5 hours (37°C, 220 rpm).

• We adjusted OD600 to 0.03 diluting the cultures in LB + Amp.

Experiment with Tecan F200

• Download Protocol

Preparation of experiment with Tecan F200 (for the following day!)

• We picked a single colony from B0015 native plate and infected 5 ml of LB + Kan.

• We incubated the inoculum overnight (37°C, 220 rpm).

August, 5th

• We received sequencing results for the first two ligation steps of ethanologenic operon and for iGEM stabs:
  • B1-13: sequence ok!
  • B2-5: sequence ok!
  • K116001: the stab actually contained K116002! moreover, sequence analysis showed an inconsistent prefix and an additional "c" at the end of nhaA promoter, but we think that the measurement system should work. We will call it with its real name ("K116002") on our freezer page.
  • K116002: the stab actually contained J33204!
  • K112405: sequence ok!
  • P0412: sequence ok!
  • I746902: sequence ok!
  • I746903: the RBS in the sequence is actually B0030 and not B0034 as documented on the Registry. Anyway, it does not corrupt the function of this brick and the rest of the sequence was ok!
  • K101017: very bad sequencing.
  • F2620MIT1: sequence ok!
  • F2620MIT2: sequence ok!

Preparation of experiment with Tecan F200

• We diluted 1:100 the overnight culture of B0015.

• We incubated the diluted culture for 5 hours (37°C, 220 rpm).

Experiment with Tecan F200

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

August, 6th

• We received Ethanol Assay Kit and Lactose Assay Kit from BioVision!

• We received sequencing results for the second ligation step of ethanologenic operon:
  • B3-1: sequence ok, but the chromatogram quality was not so good...
  • B3-5: sequence ok!
  • B4-2: sequence ok!
  • B4-4: sequence ok!

• We decided to use B3-5 and B4-2 for the following assemblies.

• We infected 4 ml of LB + Amp with the following glycerol stocks:

B1-13 (X2)	B2-5 (X2)	B3-5 (X2)
B4-2 (X2)	F2620MIT1	K112808
BOL1	R0011

• Tomorrow they will be miniprepped to prepare the assemblies of the following week!

August, 7th

• Miniprep for:

B1-13 (X2)	B2-5 (X2)	B3-5 (X2)
B4-2 (X2)	R0011	F2620MIT1
BOL1	K112808

• We stored purified DNA at -20°C and next week we will perform digestion for these 12 samples, in order to: i) finish the assembly of the ethanologenic operon, ii) re-assemble A11, which sequence analysis showed a deletion and iii) build up an inducible lysis device.

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