'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 pSB1A3 carrying 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 Fig. 2 of PCR product and gel-out of proper bands (ΔA - 250 bp).
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).

Preparation of pSB2K3 + _alpha

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).
3. Digest of purified PCR product with EcoRI and BcuI (BamHI buffer).
4. Clean-up of digested PCR product.

Preparation of pSB2K3 + _omega

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 fragment.
2. Gel electrophoresis of PCR products and gel-out of proper band (linker_omega - 350 bp).
3. Digest of purified PCR product with EcoRI and BcuI (BamHI buffer).
4. Clean-up of digested PCR product.

Preparation of BioBricks

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_omega fragment.
2. Gel electrophoresis of PCR products and gel-out of proper bands (ΔA - 250 bp, linker_alpha - 600 bp, linker_omega - 350 bp and OmpA_omega - 900 bp).
3. Digest of purified PCR products and pSB1A3 standard plasmid with EcoRI and BcuI (BamHI buffer) and pET15b+OmpA_alpha plasmid with NdeI and SacI (BamHI buffer). Dephosphorylation (CIAP) of plasmids.
4. Clean-up of digested PCR products.
5. Gel electrophoresis of digested plasmids and gel-out of proper bands (OmpA_link - 500 bp and pSB1A3 - 2200 bp).
Fig. 1. Gradient PCR (temperatures:55-75 °C)
1. Marker
2. 55 °C linker_alpha
3. 60 °C linker_alpha
4. 65 °C linker_alpha
5. 70 °C linker_alpha
Fig. 2. PCR to obtain inserts
1. Marker
2. deltaA
3 omega
4. ompA_omega