Team:Warsaw/Calendar-Main/18 September 2008

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'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.).
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

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

Fig. 3. EcoRI/BcuI digest of pSB1A3
1. Marker
2. Digested pSB1A3

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.
Fig. 4. PCR to obtain linker_alpha for reisolation from agarose gel
1. Marker
2. linker_alpha

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.

  5. 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.

Mentioned primers were used only for test purposes. During part preparation we've used another primers. Fig. 2. PCR products for reisolation from agarose gel
1. Marker
2. deltaA
3. omega
4. ompA_omega

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.
  3. Fig. 5. SacI/NdeI digest of pET15b_OmpA_omega
    1. Marker
    2. digested pSB1A3