Week from August 24th, to August 30th, 2009

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August, 24th

• We inoculated competent DH5alpha strain (gift from Bologna iGEM09 Team) in 4 ml of LB and incubated it overnight (37°C, 220 rpm).

• Miniprep for B7 (2 samples), B8 (5 samples), A14-3, A16-4 and I714891 (bacterial pellets stored at -20°C).

• Screening digestion for all the miniprepped samples:
  • B7(E-P)
  • B8(E-P)
  • A14-3(E-P)
  • A16-4(E-P)
  • I714891(E-P)

• Gel run for the digested samples.

Digestion screening.

• Gel results:
  • B7 - both screened colonies were negative (~3200bp of vector and ~3200bp of insert).
  • B8 - only B8-5 SEEMED to show the expected length for ligated plasmid (~3200bp of vector and ~4200bp of insert), but it also shows a single unexpected band...All the other 4 samples were negative (~3200bp of vector and ~3200bp of insert).
  • A14-3 - showed the expected length for ligated plasmid (2079bp of vector and ~2200bp of insert).
  • A16-4 - showed the expected length for ligated plasmid (2079bp of vector and ~1700bp of insert).
  • I714891 - showed the expected length for ligated plasmid (~2700bp of vector and ~700bp of insert).

• Comments: it seems that we have the final constitutively expressed ethanol producing operon (B8 = RBS-tetR-TT-Ptet-RBS-adhB-RBS-pdc-TT in pSB1AK3). Anyway, an unexpected band is present in this part and in its ancestor (i.e. B6). So, we decided to follow three ways:
  • sequence B8-5 and see if it is correct;
  • repeat the last two assemblies of the missing version of the ethanol producing operon: B5=B1+B4 and B7=A4+B5;
  • try to purify the expected bands from the gel through gel extraction.

• We sent purified DNA of:
  • B8-5
  • A14-3
  • A16-4
• to BMR Genomics for sequencing.

• We inoculated 8 ul of these glycerol stocks:
  • R0011
  • E0240
  • A15-2
  • B1-13
  • B4-2
• in 4 ml of LB + suitable antibiotic to grow overnight cultures (37°C, 220 rpm).

August, 25th

• DH5alpha competent cells preparation.
• We streaked a single colony LB agar plate with DH5alpha overnight liquid culture, in order to store it (tomorrow a glycerol stock for it will be prepared).

• Miniprep for:
  • R0011
  • E0240
  • A15-2
  • B1-13
  • B4-2

• Digestion for:
  • R0011(S-P)
  • E0240(X-P)
  • A15-2(E-P)
  • B1-13(E-S)
  • B4-2(E-X)
  • I714891(E-P) (the purified plasmid was stored at -20°C)

• Precipitation with sodium acetate for all the 6 samples.

• Ligations:
  • B5new = B1(E-S) + B4(E-X) in pSB1AK3
  • A17 = R0011(S-P) + E0240(X-P) in pSB1AK3
  • A18 = A15-2(E-P) + I714891(E-P) in pSB3K3
• We incubated the ligations at 16°C overnight.

Preparation of experiment with Tecan F200

• We prepared 25 ml of LB + Kan and M9 supplemented + Kan with 0%, 2.5%, 3.5% and 4.5% ethanol (weight/volume).

• We inoculated 8 ul of B0015 glycerol stock in 5 ml of LB + Kan in the morning. We let it grow during the day (37°C, 220 rpm) and after about 9 hours we diluted the culture (10 ul of B0015 bacteria in 5 ml of LB + Kan and 10 ul of B0015 bacteria in 5 ml of M9 supplemented + Kan). We incubated the diluted cultures overnight (37°C, 220 rpm).

August, 26th

• We transformed B5new, A17 and A18 overnight ligations in TOP10. We plated transformed bacteria on LB agar plates + Kan (for B5new and A18) or + Amp (for A17). We incubated B5new plate at 37°C in the morning.

• After about 10 hours we picked 8 colonies from B5new plate and infected 4 ml of LB + Kan to grow overnight cultures (37°C, 220 rpm) for screening.

• A17 and A18 plates were incubated overnight at 37°C.

• We also inoculated:
  • DH5alpha strain (4 ml of LB without antibiotic) from the single colony streaked plate;
  • E0240 (4 ml of LB + Amp).

• We received sequencing results for:
  • A15-1 - sequence ok! (long part) (actually it is not consistent with the Registry, but it is consistent with iGEM 2009 QC, in which there are some problems with K112808, even if the part should work)
  • A15-3 - sequence ok! (long part) (actually it is not consistent with the Registry, but it is consistent with iGEM 2009 QC, in which there are some problems with K112808, even if the part should work)
  • A11-2 - deletion of 20bp in lacI gene...
  • A11-3 - sequence ok!
  • B5-3 - sequence ok! (long part)
  • B6-3 - sequence ok! (long part)
  • B3-5 - sequence ok!

• COMMENTS:
  • we used A11-2 and A15-2 for A14 and A18 ligations...so A14 had to be repeated using A11-3, while we will send A15-2 to BMR for sequencing soon (we forgot to do it!).
  • B3 native stock sequence was confirmed! but we still don't know why we see two unexpected bands after B3 digestions...
  • B5-3 and B6-3 sequences were confirmed! but we still don't know why we see two unexpected bands after B5 and B6 digestions...

Preparation of experiment with Tecan F200 (for tomorrow!)

• We infected 5 ml of LB + Amp with 8 ul of B0030, A2 and T9002 glycerol stocks.

• We incubated the inocula at 37°C, 220 rpm overnight.

Preparation of experiment with Tecan F200

• We diluted 1:2000 the LB and the M9 5 ml overnight cultures of B0015 in all the 50 ml falcon tubes containing 25 ml of LB + Kan and M9 supplemented + Kan with different ethanol concentrations. Before diluting the cultures, we took 3 ml of all the media to use them as blank.

• We incubated the diluted cultures at 37°C, 220 rpm in the morning.

August, 27th

• 80% glycerol preparation.

• Glycerol stock and miniprep for the 8 cultures of B5new.

• Glycerol stock for DH5alpha strain.

• Digestion (E-P) for all the 8 DNA samples.

• Gel run for the digested samples.

No positive colonies for B5new.

• Gel results: no positive colony:'( We will repeat the ligation.

• We infected 4 ml of LB + suitable antibiotic with 8 ul of B1 and B4 glycerol stocks (to repeat "B5" ligation). We incubated these two inocula at 37°C, 220 rpm overnight.

• Colony PCR for A17 plate (6 colonies). Bacteria were inoculated in 100 ul of LB + Amp before putting them in the PCR vials, waiting for the end of the reaction.

• Electrophoresis for PCR results.

Colony PCR results on A17: colonies 1, 4, 5 and 6 seem to be correct!

• Gel results: we chose A17-4 to grow an overnight culture in LB + Amp (37°C, 220 rpm).

• We picked 7 colonies from A18 plate to inoculate 4 ml of LB + Kan. We incubated them at 37°C, 220 rpm overnight.

Preparation of experiment with Tecan F200 (for tomorrow!)

• We infected 4 ml of M9 + Amp with 8 ul of A2 and B0030 glycerol stocks.

• We incubated the inocula at 37°C, 220 rpm overnight.

Preparation of experiment with Tecan F200

• We diluted 1:100 the overnight cultures of B0030, A2 and T9002 in 5 ml of LB + Amp and in 5 ml of M9 supplemented + Amp.

• We incubated the diluted cultures at 37°C, 220 rpm for 4 hours.

Experiment with Tecan F200

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August, 28th

• We prepared a pellet for E0240, discarded supernatant (LB), dried the pellet and stored the dry pellet at -20°C, ready to be miniprepped.

• Glycerol stocks for the 7 cultures of A18 and for A17-4.

• We prepared a bacterial pellet for the 7 cultures of A18, that will be screened after having A15-2 sequencing results.

• Miniprep for A17-4, B1-13 and B4-2.

• Digestion for:
  • B1(E-S)
  • B4(E-X)

• Gel run for B1(E-S) (1 ul for check) and for B4(E-X) (all the volume). Gel cut/purification for B4(E-X); precipitation with sodium acetate for B1(E-S).

• Ligation: B5new2 = B1(E-S) + B4 (E-X) in pSB1AK3. We incubated the ligation overnight at 16°C.

• We sent A17-4 purified DNA to BMR Genomics for sequencing.

Preparation of experiment with Tecan F200

• We diluted 1:10 the overnight cultures of B0030 and A2 in 5 ml of M9 supplemented + Amp.

• We incubated the diluted cultures at 37°C, 220 rpm for 4 hours.

August, 29th

• We inactivated T4 ligase (65°C for 10 min) and put the ligation at -20°C.

August, 30th

• We transformed B5new2 ligation, stored at -20°C with a 1:60 dilution. We incubated the plated bacteria at 37°C overnight.

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