Composite

Part:BBa_M36631:Experience

Designed by: Benjamin Yeh, Jodie Sheffels, Ali Hoffer   Group: Stanford BIOE44 - S11   (2015-10-24)

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Stanford Location

Part name: SHY_AFP_94 (*The label was misprinted as "SHY_APF_94"*)

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 #: 232612

Organism: E. coli

Device type: actuator

Glycerol stock barcode #: 0133021813

Box label: BIOE44 F15 Box 2

Performance data

Protein Production Dose Response Curve

Afp94 gfp-dose-response.png

Error bars represent standard error.

AFP94 protein was successfully produced in E. coli, with protein expression fully induced at 1mM IPTG.


Protein Chitin Binding Activity

Afp94 gfp-activity.png

To test the chitin sensitivity of the AFP94 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_M36631

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