Difference between revisions of "Part:BBa M36630:Experience"
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Plasmid vector from DNA2.0 into which this part was inserted: pD441-CC | Plasmid vector from DNA2.0 into which this part was inserted: pD441-CC | ||
- IPTG-inducible promoter | - IPTG-inducible promoter | ||
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- CometGFP-fusion reporter | - CometGFP-fusion reporter | ||
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- Kanamycin resistance selection marker | - Kanamycin resistance selection marker | ||
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- Strong RBS | - Strong RBS | ||
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- Origin of replication with high copy number | - Origin of replication with high copy number | ||
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DNA2.0 Gene #: 231821 | DNA2.0 Gene #: 231821 | ||
Revision as of 10:43, 7 December 2015
This experience page is provided so that any user may enter their experience using this part.
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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 #: 231821
Organism: E. coli
Device type: actuator
Glycerol stock barcode #: 0133018595
Box label: BIOE44 F15
Performance data
Protein Production Dose Response Curve
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
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.
Applications of BBa_M36630
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