Difference between revisions of "Part:BBa K103017:Experience"
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===Applications of BBa_K103017===
 
===Applications of BBa_K103017===
  
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===User Reviews===
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==='Hunter and prey' selection system===
 
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| style='border:1px solid gray; border-collapse: collapse;' | 0.09
 
| style='border:1px solid gray; border-collapse: collapse;' | 0.09
 
| style='border:1px solid gray; border-collapse: collapse;' | 0.685
 
| style='border:1px solid gray; border-collapse: collapse;' | 0.685
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| style='border:1px solid gray; border-collapse: collapse;' | 1.378
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! style='border:1px solid gray; border-collapse: collapse;' | 1.378
 
| style='border:1px solid gray; border-collapse: collapse;' | 1.078
 
| style='border:1px solid gray; border-collapse: collapse;' | 1.078
 
| style='border:1px solid gray; border-collapse: collapse;' | 0.977
 
| style='border:1px solid gray; border-collapse: collapse;' | 0.977
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We have used 100 ug/ml ampicillin and 0.25 uM IPTG for further experiments.
  
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To check if our hunter expression devices work we have tested growth of hunter expressing strains in liquid LB + Ampicillin + IPTG suplemented with His_omega_A (compatible with OmpA_alpha_Z hunter) and His_omega_Z (compatible with OmpA_alpha_A) prey proteins.
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Following results were obtained:
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 +
{| style='border:1px solid gray; border-collapse: collapse;'
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|-
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! style='border:1px solid gray; border-collapse: collapse;' | Hunter variant
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! style='border:1px solid gray; border-collapse: collapse;' | Prey variant
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! style='border:1px solid gray; border-collapse: collapse;' | Growth with prey
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! style='border:1px solid gray; border-collapse: collapse;' | Growth without prey
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|-
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! style='border:1px solid gray; border-collapse: collapse;' | OmpA_alpha
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| style='border:1px solid gray; border-collapse: collapse;' | His_Z_omega
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| style='border:1px solid gray; border-collapse: collapse;' | -
 +
| style='border:1px solid gray; border-collapse: collapse;' | -
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|-
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! style='border:1px solid gray; border-collapse: collapse;' | OmpA_alpha
 +
| style='border:1px solid gray; border-collapse: collapse;' | His_Z_alpha
 +
| style='border:1px solid gray; border-collapse: collapse;' | -
 +
| style='border:1px solid gray; border-collapse: collapse;' | -
 +
|-
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! style='border:1px solid gray; border-collapse: collapse;' | Omp_alpha_A
 +
| style='border:1px solid gray; border-collapse: collapse;' | His_Z_omega
 +
| style='border:1px solid gray; border-collapse: collapse;' | +
 +
| style='border:1px solid gray; border-collapse: collapse;' | -
 +
|-
 +
! style='border:1px solid gray; border-collapse: collapse;' | Omp_alpha_A
 +
| style='border:1px solid gray; border-collapse: collapse;' | His_Z_alpha
 +
| style='border:1px solid gray; border-collapse: collapse;' | -
 +
| style='border:1px solid gray; border-collapse: collapse;' | -
 +
|-
 +
! style='border:1px solid gray; border-collapse: collapse;' | Omp_alpha_Z
 +
| style='border:1px solid gray; border-collapse: collapse;' | His_Z_omega
 +
| style='border:1px solid gray; border-collapse: collapse;' | +
 +
| style='border:1px solid gray; border-collapse: collapse;' | -
 +
|-
 +
! style='border:1px solid gray; border-collapse: collapse;' | Omp_alpha_Z
 +
| style='border:1px solid gray; border-collapse: collapse;' | His_Z_alpha
 +
| style='border:1px solid gray; border-collapse: collapse;' | -
 +
| style='border:1px solid gray; border-collapse: collapse;' | -
 +
|-
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! style='border:1px solid gray; border-collapse: collapse;' | Omp_alpha_A_omega
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| style='border:1px solid gray; border-collapse: collapse;' | His_Z_omega
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| style='border:1px solid gray; border-collapse: collapse;' | +
 +
| style='border:1px solid gray; border-collapse: collapse;' | +
 +
|-
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! style='border:1px solid gray; border-collapse: collapse;' | Omp_alpha_A_omega
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| style='border:1px solid gray; border-collapse: collapse;' | His_Z_alpha
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| style='border:1px solid gray; border-collapse: collapse;' | +
 +
| style='border:1px solid gray; border-collapse: collapse;' | +
 +
|-
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|}
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 +
Addition of compatible prey to the culture medium makes hunter expressing cells survive. Unfortunately there are also false positives (Omp_alpha_Z grows on medium containing His_Z_omega). This may be caused by ability of Z<sub>spa-1</sub> protein to form autoaggregates.
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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.
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Conclusion: hunter and prey system works as expected allowing efficient selection of strain expressing the strongest interactor of a given protein.
  
 
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Revision as of 12:34, 25 October 2008

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.

Applications of BBa_K103017

'Hunter and prey' selection system

UNIQ6e42acc56450c9f7-partinfo-00000000-QINU

•••••

Keton

We have used this part in fusion with BBa_K103003 and BBa_K103004 to create hunter expression devices:

To check optimal IPTG and ampicilin concentrations we have used OmpA_alpha_A_omega positive control device which should make cells ampicilin resistant. We have obtained following results:

OD600 of Top10 cultures expressing OmpA_alpha_A_omega
Ampicilin concentration (ug /ml) IPTG Concentration (uM)
0 0.1 0.25 0.5 0.75 1
25 1.558 1.469 1.587 1.49 1.566 1.311
50 1.425 1.435 1.524 1.055 0.920 0.935
75 1.09 0.989 1.447 0.971 0.951 0.992
100 0.09 0.685 1.378 1.078 0.977 0.992

We have used 100 ug/ml ampicillin and 0.25 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_omega_A (compatible with OmpA_alpha_Z hunter) and His_omega_Z (compatible with OmpA_alpha_A) prey proteins.

Following results were obtained:

Hunter variant Prey variant Growth with prey Growth without prey
OmpA_alpha His_Z_omega - -
OmpA_alpha His_Z_alpha - -
Omp_alpha_A His_Z_omega + -
Omp_alpha_A His_Z_alpha - -
Omp_alpha_Z His_Z_omega + -
Omp_alpha_Z His_Z_alpha - -
Omp_alpha_A_omega His_Z_omega + +
Omp_alpha_A_omega His_Z_alpha + +

Addition of compatible prey to the culture medium makes hunter expressing cells survive. Unfortunately there are also false positives (Omp_alpha_Z grows on medium containing His_Z_omega). 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.

UNIQ6e42acc56450c9f7-partinfo-00000002-QINU