Part:BBa_K103018:Experience

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

'Hunter and prey' selection system

UNIQfbb853873a0a4d64-partinfo-00000000-QINU

••••• Keton

We have used this part in fusion with BBa_K103003 and BBa_K103004 to create hunter expression devices: OmpA_omega_A (compatible prey: His_alpha_Z - fusion of BBa_K103019 with BBa_K103004) OmpA_omega_Z (compatible prey: His_alpha_A - fusion of BBa_K103019 with BBa_K103003) OmpA_omega negative control device without hunter BBa_K103018 OmpA_omega_A_alpha positive control device containing both beta-lactamase halves BBa_K103018 in fusion with BBa_K103003 and BBa_K103008 To check optimal IPTG and ampicilin concentrations we have used OmpA_omega_A_alpha positive control device which should make cells ampicilin resistant. We have obtained following results: OD600 of Top10 cultures expressing OmpA_omega_A_alpha Ampicilin concentration (ug /ml) IPTG Concentration (uM) 0 0.1 0.25 0.5 0.75 1 25 1.054 1.154 1.051 0.99 1.096 0.896 50 0.94 0.891 1.123 0.847 0.924 0,81 75 0.5 0.63 0.743 0.782 0.631 0.64 100 0.02 0.396 0.563 0.678 0.602 0.611 We have used 100 ug/ml ampicillin and 0.5 uM IPTG for further experiments. To check if our hunter expression devices work we have tested growth of hunter expressing strains in liquid LB + Ampicillin + IPTG suplemented with His_alpha_A (compatible with OmpA_omega_Z hunter) and His_alpha_Z (compatible with OmpA_omega_A) prey proteins. Following results were obtained: Hunter variant Prey variant Growth with prey Growth without prey OmpA_omega His_alpha_Z - - OmpA_omega His_omega_Z - - Omp_omega_A His_alpha_Z + - Omp_omega_A His_omega_Z - - Omp_omega_Z His_alpha_Z + - Omp_omega_Z His_omega_Z - - Omp_omega_A_alpha His_omega_Z + + Omp_omega_A_alpha His_alpha_Z + + Addition of compatible prey to the culture medium makes hunter expressing cells survive. Unfortunately there are also false positives (Omp_omega_Z grows on medium containing His_omega_Z). This may be caused by ability of Zspa-1 protein to form autoaggregates. Concurrence tests were the next step in testing hunter and prey system. Two or more bacterial strains were mixed and added to medium with prey. The culture was incubated overnight and plasmid miniprep was prepared. The strains which survived were determined using restriction digests and PCR. Results were very good: in all cases strains encoding strongest interacting hunters always dominate the culture and are detectable both by PCR and restriction digests. The DNA yield from other strains was very small and can be detected only by PCR. Conclusion: hunter and prey system works as expected allowing efficient selection of strain expressing the strongest interactor of a given protein.

;

UNIQfbb853873a0a4d64-partinfo-00000002-QINU