'Hunter and prey' system tests: Competition tests

Weronika

Inoculation of pACYC177+OmpA_A_alpha, pACYC177+OmpA_Z_omega, pACYC177+OmpA_A_omega and pACYC177+OmpA_Z_alpha + induction using 0.25mM iPTG

MutD₅ testing

Piotr

Inoculation of mutD5 into 100 ml of LB and rendering them chemocompetent. Electroporation with pACYC177+OmpA_Z_alpha, pACYC177+OmpA_Z_omega, pACYC177+OmpA_A_omega and pACYC177+OmpA_omega_ΔA.

Optimisation of primers for preparation of parts

Michał K.

1. Repetition of PCR on pACYC177+OmpA_omega_ΔA_alpha plasmid using LinLSXNE and AlphaPSpe primers (elongation length 60s) to obtain linker_alpha (BBa_K103009) fragment. Reaction was carried out with 25 cycles and in temperature gradient from 55 to 75 °C (four reactions).
2. Gel electrophoresis of PCR products (Fig. 1.).

Preparation of ΔA (BBa_K103003)

Michał K.

1. PCR on pACYC177+OmpA_omega_ΔA_alpha plasmid using AL_BNXNE and APSacSpe primers (annealing temperature 58 °C; elongation length 45s) to obtain ΔA fragment.
2. Gel electrophoresis of PCR product and gel-out of proper bands (ΔA - 250 bp). Fig. 2.
3. Digest of purified PCR product and pSB1A3 standard plasmid with EcoRI and BcuI (BamHI buffer). Dephosphorylation (CIAP) of plasmid.
4. Clean-up of digested PCR product.
5. Gel electrophoresis of digested plasmid and gel-out of proper band (pSB1A3 - 2200 bp).Fig. 3.

Fig. 2. PCR to obtain inserts
1. Marker
2. DeltaA
3. Omega
4. OmpA_omega

Preparation of linker_alpha (BBa_K103009)

Michał K.

1. PCR on pACYC177+OmpA_omega_ΔA_alpha plasmid using LinLSXNE and AlphaPSpe primers (annealing temperature 65 °C; elongation length 60s) to obtain linker_alpha (BBa_K103009) fragment.
2. Gel electrophoresis of PCR product and gel-out of proper bands (linker_alpha - 600 bp).Fig. 4.
3. Digest of purified PCR product with EcoRI and BcuI (BamHI buffer).
4. Clean-up of digested PCR product.

Preparation of linker_omega (BBa_K103013)

Michał K.

1. PCR on pACYC177 + OmpA_omega plasmid using LinLSXNE and OmegPSpe primers (annealing temperature 55 °C; elongation length 60s) to obtain linker_omega (BBa_K103013) fragment.
2. Gel electrophoresis of PCR products and gel-out of proper band (linker_omega - 350 bp). Fig. 2.
3. Digest of purified PCR product with EcoRI and BcuI (BamHI buffer).
4. Clean-up of digested PCR product.


Fig. 2. PCR to obtain inserts
1. Marker
2. DeltaA
3. Omega
4. OmpA_omega

Preparation of OmpA-linker-omega-linker (BBa_K103016)

Michał K.

1. PCR on pACYC177+OmpA_omega_ΔA_alpha plasmid using OmpLNXNE and LinP_BS primers (annealing temperature 58 °C; elongation length 90s) to obtain OmpA-linker-omega-linker (BBa_K103016) fragment.
2. Gel electrophoresis of PCR products and gel-out of proper bands (OmpA-linker-omega-linker - 900 bp). Fig. 2.
3. Digest of purified PCR product with EcoRI and BcuI (BamHI buffer).
4. Clean-up of digested PCR products.

Preparation of OmpA-linker (BBa_K103006)

Michał K.

1. Digest of pET15b+OmpA_alpha plasmid with NdeI and SacI (BamHI buffer). Dephosphorylation (CIAP) of plasmid.
2. Gel electrophoresis of digested plasmid and gel-out of proper band (OmpA_linker - 500 bp). Fig. 5.
Fig. 5. SacI/NdeI digest of pET15b_OmpA_omega
1. Marker
2. digested pSB1A3