Composite

Part:BBa_M36630:Experience

Designed by: Benjamin Yeh, Jodie Sheffels, Ali Hoffer   Group: Stanford BIOE44 - S11   (2015-10-24)
Revision as of 10:47, 7 December 2015 by Bentyeh (Talk | contribs)

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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

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.

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