Visit in US Embassy

Visas have been accorded to the whole team.

Preparation of alpha_linker under PT7 (BBa_K103019)

Michał K., Piotr

1. Clean-up of overnight digest reaction.
2. Digest of pSB1A3 carrying ΔA (BBa_K103003) with EcoRI and SacI (BamHI buffer), dephosphorylation with CIAP
3. Gel electrophoresis and gel-out of proper band - 2200 bp. Fig. 1.
4. Ligation of digested pSB1A3 with alpha_linker under PT7 (BBa_K103019) fragment (1 hr).
5. Transformation of TOP10 with above ligation.
6. Tranformants plating on LB with ampicillin.

Fig. 1.PCR to obtain alpha, PCR to obtain ara_pbad_aid, digest of pACYC177+OmpA_omega_DeltaA_alpha
1. Marker
2. PCR to obtain alpha_linker under PT7 (BBa_K103019)
3. PCR to obtain AID under pBAD/araC (BBa_K103002)
4. EcoRI/BcuI digest of pACYC177+OmpA_omega_DeltaA_alpha

Preparation of omega_linker under PT7 (BBa_K103020)

Michał K., Piotr

1. Ligation of digested pSB2K3 vector from (30 September) with omega_linker under PT7 (BBa_K103020) fragment (1 hr).
2. Transformation of TOP10 with above ligation.
3. Tranformants plating on LB with kanamycin.

Preparation of AID under pBAD/araC (BBa_K103002)

Michał K., Piotr

1. Isolation of plasmid from culture inoculated on previous day - pMPMT5+AID (with removed EcoRI site).
2. Control digest of isolated plasmids with EcoRI and BamHI (BamHI buffer) - proper clones found.
3. Temperature gradient PCR on pMPMT5+AID (with removed EcoRI site) plasmid using AraCl and AIDP_HindSpeNotPst primers (annealing temperature 40 - 60 °C; elongation length 2.30 min) to optimize conditions for obtaining AID under pBAD/araC (BBa_K103002).
4. Gel electrophoresis. Best annealing temperature (45 °C) chosen. Fig. 2.
5. PCR on pMPMT5+AID (with removed EcoRI site) plasmid using AraCl and AIDP_HindSpeNotPst primers (annealing temperature 45 °C; elongation length 2.30 min) to obtain AID under pBAD/araC (BBa_K103002)fragment.
6. Gel electrophoresis and gel-out of proper band 1800 bp. Fig. 1.
7. Digest of isolated PCR product with XbaI and PstI (Tango buffer).
8. Clean-up of digested PCR product.

Fig. 1.PCR to obtain alpha, PCR to obtain ara_pbad_aid, digest of pACYC177+OmpA_omega_DeltaA_alpha
1. Marker
2. PCR to obtain alpha_linker under PT7 (BBa_K103019)
3. PCR to obtain AID under pBAD/araC (BBa_K103002)
4. EcoRI/BcuI digest of pACYC177+OmpA_omega_DeltaA_alpha

Preparation of OmpA_linker_alpha_linker under Plac (BBa_K103017)

Michał K.

1. PCR on pACYC177+OmpA_omega_ΔA_alpha plasmid using AlphaL+SacI and AlphaP+link10+homo2 primers (annealing temperature 58 °C; elongation length 60s) to obtain alpha fragment.
2. Gel electrophoresis and gel-out of proper band 600 bp. Fig. 1.