Week from June 15th, to June 21st, 2009

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June, 15th

• This week we wanted to complete the assembly of an aTc sensor and to test if lacZ works. Sequence analysis cannot be efficiently performed to I732017 or to A3 because of lacZ coding sequence length (>3 Kb), so we decided to check lacZ integrity assembling a constitutive promoter upstream of A3 and testing the ligation on X-Gal plates.

• We already had J23100, J23101, A3 and A4 at -20°C. Digestions:

• We ran/cut a 1% gel for J23100(S-P), A3(E-X) and A4(X-P). All the desired bands had the expected sizes and had been purified from agarose gel. Unlikely we had a low yield for A4(X-P) extraction...

• We ran a 2% gel to isolate J23101(E-S) insert, but unfortunately we could not see any band at the expected size of 35 bp...

• We planned not to perform any ligation: unlucky day for them!:(
• NOTE: we did not re-try to extract J23101(E-S) because it is too small.
  

Our A4 ligation is composed by RBS-tetR-TT-Ptet , so it can be considered as a constitutive promoter (Ptet) with an additional "non functional" part (RBS-tetR-TT). So it can be easily used as an insert for assembly because of its 900 bp size, that allows it to be efficiently isolated and extracted from a gel!

• For this reason, we infected 5 ml of LB + Amp with 15 ul of A4 glycerol stock (X4 falcon tubes). The following day we plan to cut A4(E-S) (X2) to use it as a constitutive promoter for A3(E-X), while we plan to cut A4(X-P) (X2) to ligate it to J23100 to constitute an aTc->PoPS inducible device. We performed all the inocula X2 because we wanted to be sure to have a sufficient yield for ligations.

• We stored J23100(S-P) and A3(E-X) at -20°C.

June, 16th

• Miniprep for A4 (X4 overnight cultures).

• Digestions:

• Gel run/cut; gel extraction.

• Ligations:
  • A5 = A4(E-S) + A3(E-X) in pSB1AK3
  • A6 = J23100(S-P) + A4(X-P) in J61002 without RFP protein generator

• We incubated the two ligation reactions overnight at 16°C.

June, 17th

• We prepared three X-Gal plates (of LB + Amp).

• We transformed the two ligations (1 ul) in TOP10 E. coli.

• We plated A6 transformed bacteria + SOC in a non-X-Gal plate.
• We plated A5 transformed bacteria + SOC in two X-Gal plates: we plated 100 ul in the first plate (called A5) and 200 ul in the second plate (called A5bis) in order to check for blue colonies the following day.

• We also plated 20 ul of E0240 glycerol stock in the third prepared X-Gal plate, in order to have a negative control to check if TOP10 E. coli actually don't express beta-galactosidase.

• Expected results:
  • Plates A5 and A5bis should show blue colonies;
  • Plate E0240 should remain colorless.

June, 18th

• Plate results:
  • A5 showed blue colonies;
  • A5bis showed blue colonies;
  • E0240 did not show any blue colony.

• So, we can say that TOP10 E. coli don't express any beta-galactosidase and our lacZ gene works!

• We decided to perform the X-Gal test on A3, which is the promoterless RBS-lacZ-TT in pSB1AK3:
  • we prepared another X-Gal plate (LB + Amp),
  • we mixed 20 ul of A3 glycerol stock with 100 ul of SOC medium,
  • we plated the diluted A3 culture and incubated the plate overnight at 37°C.

• Colony PCR for A6 plate: we screened six colonies. We let the picked colonies grow in 1 ml of LB + Amp, 37°C, 220 rpm, waiting for the screening results.

• We had a problem with the thermal cycler, it gave an error after about 20 min of the reaction and we had to restart it. At the end of the reaction we noticed that the cap of A6-2 vial had been melted and almost all the reaction evaporated...What a beautiful PCR! isn't it? @_@(...)

• Electrophoresis for the PCR vials.

• Gel results: the almost evaporated reaction (A6-2) did not show any amplicon (lane 8). All the other reactions showed the amplicon with the expected size (~1100 bp).

• We chose the 4th colony (A6-4): we filled A6-4 for a total volume of 5 ml and let it grow overnight at 37°C, 220 rpm.

Preparation of experiment with Tecan F200

• We picked two colonies from E0240 overnight plate and infected two falcon tubes containing 5 ml of LB + Amp. We incubated the inocula at 37°C, 220 rpm for 2 hours before the test.

Experiment with Tecan F200

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

June, 19th

June, 20th

June, 21st