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

Revision as of 10:40, 7 December 2015

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Stanford Location

Part name: SHY_AFP_51

Plasmid vector from DNA2.0: pD441-CC (this part was, BBa_M36630 was inserted into this vector for testing) - 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

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

User Reviews

UNIQe2de19f9810efe84-partinfo-00000000-QINU UNIQe2de19f9810efe84-partinfo-00000001-QINU

@@ Line 30: / Line 30: @@
 [[File:Afp51_gfp_dose_response.png|720px]]
-AFP51 protein was successfully produced in E. coli, with protein expression fully induced at 1mM IPTG.
+AFP51 protein was successfully produced in ''E. coli'', with protein expression fully induced at 1mM IPTG.
 '''Protein Chitin Binding Activity'''