Difference between revisions of "Part:BBa M36630:Experience"

 
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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 
This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 
how you used this part and how it worked out.
 
how you used this part and how it worked out.
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===Stanford Location===
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Part name: SHY_AFP_51
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Plasmid vector from DNA2.0 into which this part was inserted: pD441-CC
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- IPTG-inducible promoter
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- CometGFP-fusion reporter
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- Kanamycin resistance selection marker
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- Strong RBS
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- Origin of replication with high copy number
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DNA2.0 Gene #: 232611
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Organism: ''E. coli''
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Device type: actuator
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Glycerol stock barcode #: 0133010032
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Box label: BIOE44 F15 Box 2
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===Performance data===
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'''Protein Production Dose Response Curve'''
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[[File:Afp51_gfp_dose_response.png|720px]]
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Error bars represent standard error.
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AFP51 protein was successfully produced in ''E. coli'', with protein expression fully induced at 1mM IPTG.
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'''Protein Chitin Binding Activity'''
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[[File:Afp51_gfp-activity.png|720px]]
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To test the chitin sensitivity of the AFP51 protein, we incubated various concentrations of AFP with a constant amount of chitin. We designed this assay based on a preliminary experiment (data not shown) that suggested that the fluorescence of a lysate solution containing the GFP-tagged AFP protein could be reduced by incubation with chitin beads that were then separated from the lysate (because the proteins bound to the beads).
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Surprisingly, all of our experimental samples showed increased fluorescence after incubation with chitin beads. This was contrary to what we had expected to observe. That there was no decrease in fluorescence suggests that AFP remained in the lysate, instead of binding to the beads. However, the marked increase in fluorescence is more difficult to explain. Not only did fluorescence increase in every trial, but it did so in a proportional and consistent manner. Samples with higher starting fluorescence showed greater increases in fluorescence after incubation with chitin beads. We suspect that GFP maturation may be responsible for the increase in fluorescence over time. Additionally, the GFP-fusion protein may have inhibited the affinity of the small AFP protein for chitin.
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In short, our part was produced, but it did not function as expected.
  
 
===Applications of BBa_M36630===
 
===Applications of BBa_M36630===

Latest revision as of 11:00, 7 December 2015

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.

Stanford Location

Part name: SHY_AFP_51

Plasmid vector from DNA2.0 into which this part was inserted: pD441-CC

- IPTG-inducible promoter

- CometGFP-fusion reporter

- Kanamycin resistance selection marker

- Strong RBS

- Origin of replication with high copy number


DNA2.0 Gene #: 232611

Organism: E. coli

Device type: actuator

Glycerol stock barcode #: 0133010032

Box label: BIOE44 F15 Box 2

Performance data

Protein Production Dose Response Curve

Afp51 gfp dose response.png

Error bars represent standard error.

AFP51 protein was successfully produced in E. coli, with protein expression fully induced at 1mM IPTG.


Protein Chitin Binding Activity

Afp51 gfp-activity.png

To test the chitin sensitivity of the AFP51 protein, we incubated various concentrations of AFP with a constant amount of chitin. We designed this assay based on a preliminary experiment (data not shown) that suggested that the fluorescence of a lysate solution containing the GFP-tagged AFP protein could be reduced by incubation with chitin beads that were then separated from the lysate (because the proteins bound to the beads).

Surprisingly, all of our experimental samples showed increased fluorescence after incubation with chitin beads. This was contrary to what we had expected to observe. That there was no decrease in fluorescence suggests that AFP remained in the lysate, instead of binding to the beads. However, the marked increase in fluorescence is more difficult to explain. Not only did fluorescence increase in every trial, but it did so in a proportional and consistent manner. Samples with higher starting fluorescence showed greater increases in fluorescence after incubation with chitin beads. We suspect that GFP maturation may be responsible for the increase in fluorescence over time. Additionally, the GFP-fusion protein may have inhibited the affinity of the small AFP protein for chitin.

In short, our part was produced, but it did not function as expected.

Applications of BBa_M36630

User Reviews

UNIQff93a14038551f5e-partinfo-00000000-QINU UNIQff93a14038551f5e-partinfo-00000001-QINU