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Team:UNIPV-Pavia/Notebook/Week3Jun
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*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. | *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: |
| + | {|cellpadding="20" | ||
| + | |J23100(S-P) | ||
| + | |J23101(E-S) | ||
| + | |- | ||
| + | |A3(E-X) | ||
| + | |A4(X-P) | ||
| + | |} | ||
| + | |||
| + | *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.<br> | ||
| + | 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 used as an insert for BioBrick Standard Assembly because of its 900 bp size, that allows it to be easily 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. | ||
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Revision as of 14:40, 21 June 2009
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Week from June 15th, to June 21st, 2009
Previous Week
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Next Week
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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:
| J23100(S-P) | J23101(E-S) |
| A3(E-X) | A4(X-P) |
- 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 used as an insert for BioBrick Standard Assembly because of its 900 bp size, that allows it to be easily 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.
June, 16th
June, 17th
Trasformation of Ligations
A5: BBa_Q0040 with BBa_I732017 (RBS-TETR-PTET-RBS-LACZ-TT)
A6: BBa_J23100 with BBa_Q0040 (PCON-RBS-TETR-TT-PTET )
- 1 ul A5 in Top10 Competent
- 1 ul A6 in Top10 Competent
Mix 80 ul of SOC with 20 ul X-GAL (40 mg/ml) and infect LB+Amp Agar plates.
- Add 250 ul SOC in 50 ul A5+ Top10
- Add 250 ul SOC in 50 ul A6+ Top10
1 hr incubation at 37°C (falcons with shaking 220 rpm)
Infect:
- X-GAL plate with 200 ul A5
- X-GAL plate with 100 ul A5
- Agar plate with 200 ul A6
- X-GAL plate with 20 ul of BBa_E0240 from glycerol stock (negative control).
Expected results:
- Plates 1 and 2 should develop blue colonies
- Plate 4 should remain colorless
16 hr incubation at 37°C.
June, 18th
Experiment with Tecan F200
- Description
- Purpose:
- Materials & Methods
- Protocol
- Results
June, 19th
June, 20th
June, 21st
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