Week from June 22nd, to June 28th, 2009

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June, 22nd

• This week we planned to ligate the GFP protein generator under the control of Ptet in A4 and A6 constructs, in order to have:
  • an aTc->GFP measurement system (A6-E0240 = J23100-RBS-tetR-TT-Ptet-RBS-GFP-TT) and
  • a control construct that should always express GFP(A4-E0240 = RBS-tetR-TT-Ptet-RBS-GFP-TT)
• Moreover, we planned to begin the construction of a lysis actuator with K117000 (=celB) BioBrick and also a test construct with J23118 to assay GFP expression.

• We infected 5 ml of LB + Amp with 10 ul of these glycerol stocks (to prepare samples for this week's ligations):

• We incubated the 5 ml cultures overnight at 37°C, 220 rpm.

• We transformed BOL1 plasmid in TOP10 and plated transformed bacteria in a LB + Amp agar plate. We incubated the plate overnight at 37°C.

• Wiki updating.

• We ordered pdc and adhB coding sequences from Zymomonas mobilis to Mr Gene. They had the following specifications:
  • the sequences were taken from the online data banks, considering the most recent entries;
  • the organism of interest was Z. mobilis CP4 (ATCC 31821);
  • the sequences were codon-optimized for E. coli;
  • prefix and suffix sequences were added, considering that both genes start with ATG;
  • an additional stop codon (TAA) was addad at the end of the coding sequence, just before the suffix, in order to have a double stop codon;
  • restriction sites EcoRI, XbaI, SpeI, PstI and NotI had to be avoided during codon optimization (while the original coding sequences didn't have any of them).

June, 23rd

• BOL1 plate showed colonies! We picked a colony from BOL1 plate and infected 1 ml of LB + Amp. We incubated the inoculum at 37°C, 220 rpm for 5 and 1/2 hours.

• Glycerol stock for the grown culture of BOL1.

• We re-filled the remaining 250 ul of BOL1 culture with 5 ml of LB + Amp and incubated the culture overnight at 37°C, 220 rpm. The next day it will be miniprepped and sent to BMR for sequencing!

• Miniprep for: E0240 (5 samples), A4, A6, B0015, J23118 and K117000
• Digestions:

• The bands were cut from the gel and the following ligations were performed:
  • A7 = J23118(S-P) + E0240(X-P)
  • A8 = A4(S-P) + E0240(X-P)
  • A9 = A6(S-P) + E0240(X-P)
  • A10 = K117000(E-S) + TT(E-X)

• The ligation reactions were incubated overnight at 16°C.

June, 24th

• Miniprep for BOL1. We also sent the purified plasmid to BMR Genomics for sequencing.
• We resuspended R0011 (=Plac) and K112808 (=lysis actuator from T4 phage) from the iGEM plates with 15 ul of RNAse free water.
• Trasformations in TOP10 bacteria were done for: A7, A8, A9, A10, K112808 and R0011. We plated the transformed bacteria and incubated the plates overnight at 37°C.
• Team meeting
• We received sequencing results for:
  • K131009 - MIT sequencing was confirmed: celB had a point mutation which changes 1 amino acid L->M, while both prefix and suffix had a nucleotide deletion (anyway, the restriction sites were not corrupted). We will document it on K131009 page, but by now we don't know if it is a good idea to use this BioBrick...
  • A3 - correct! (long part, but lacZ had already been successfully tested)
  • A4 - correct!
  • A5? - correct! (long part, but lacZ had already been successfully tested), we were very lucky: the colony had been chosen randomly from the A5 plate:):) Now "A5?" will be called "A5"!
  • A6 - correct!

June, 25th

• All the overnight plates showed colonies!

• We picked a colony from R0011 and K112808 plates to infect 1 ml of LB + Amp. We incubated the two inocula at 37°C, 220 rpm for 5 and 1/2 hours.

• Glycerol stocks for R0011 and K112808. The remaining 250 ul of R0011 grown culture had been re-filled with 5 ml of LB + Amp to grow an overnight culture (37°C, 220 rpm).

• We also infected 5 ml of LB + Amp with 10 ul of BOL1 glycerol stock to grow an overnight culture (37°C, 220 rpm).

• Colony PCR for:
  • A7 - 3 colonies (few colonies because we picked non red coloured colonies)
  • A8 - 8 colonies (important ligation!)
  • A9 - 12 colonies (important ligation!)
  • A10 - 3 colonies (it's quite unuseful to screen this ligation, because probably we are not able to discriminate the positive and the negative transformants)

• The picked colonies used in the PCR were inoculated in 1 ml of LB + Amp and incubated at 37°C, 220 rpm, waiting for the gel results.

• We performed two different PCR programs:
  • one for A7 (expected size of positive plasmid: ~1 Kb) and A10 (expected size of positive plasmid: ~600 bp), with 3 min elongation time
  • and a different one for A8 and A9 (expected size of positive plasmids: ~2 Kb for both).

• Electrophoresis for the resulting reactions.

Colony PCR on A7, A8, A9, A10: we chose the 1st screened colony for A7 (A7-1), while we did not choose any colony for the other ligations because they did not look correct.

• Gel results:
  • A7 - all colonies were good, we kept A7-1 to store a glycerol stock.
  • A8, A9 and A10 unfortunately showed extra bands. So, we didn't keep any colony for them.
  • Blank reaction also showed a contaminant band.

• We planned:
  • not to care about A10 at the moment because the priority was to test aTc sensing device with A8 and A9;
  • to re-examinate the gel results to explain the lengths of the extra bands (on Monday);
  • to re-transform the ligations (stored at -20°C) because probably more than one plasmid had been incorporated in the screened colonies...

Preparation of experiment with Tecan F200

• We infected 5 ml of LB + Amp with 10 ul of:
  • T9002 (the following day it will be diluted and induced with different concentrations of 3OC6-HSL)
  • E0240 (negative control)
  • A1 (positive control)
• glycerol stocks. We incubated the inocula overnight at 37°C, 220 rpm.

June, 26th

• Miniprep for R0011 and BOL1. The purified plasmids were stored at -20°C ready to be cut!

*We diluted 1:20 the A8 and A9 ligations, which were stored at -20°C, and transformed 1 ul of the dilution in TOP10 E. coli. We plated the transformed bacteria on LB + Amp agar plates and incubated the two plates at 37°C overnight.

Susanna resuspending pellets during miniprep.

Preparation of experiment with Tecan F200

• We diluted 1:1000 the overnight cultures of:
  • T9002 (24 different 5 ml cultures)
  • E0240 (one 5 ml culture)
  • A1 (one 5 ml culture)

• We induced the 24 cultures of T9002 with eigth different concentrations of 3OC6-HSL (three 5 ml cultures for each concentration): 0 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1 uM, 10 uM, 100 uM.

• We incubated the cultures overnight at 37°C, 220 rpm for 3 hours.

Experiment with Tecan F200

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

June, 27th

• The two overnight plates showed colonies. We put the two grown plates at +4°C: next week they will be screened!

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