Results

Team Parts Sandbox

Name	Type	Description	Designer	Length	Favorite Part
[http://parts.igem.org/Part:BBa_K1419000 BBa_K1419000]	Device	Arabinose inducible lysis casette	Zak Stinson	3002
[http://parts.igem.org/Part:BBa_K1419001 BBa_K1419001]	RNA Part	Arabinose inducible lysis casette with RNA-IN	Zak Stinson	32
[http://parts.igem.org/Part:BBa_K1419002 BBa_K1419002]	Device	RNA-OUT component for an Arabinose inducible lysis casette	Harland Brandon	117****#$*#$
[http://parts.igem.org/Part:BBa_K1419003 BBa_K1419003]	Device	RNA-OUT component for a Universal Regulation System	Harland Brandon	117****#$*#$
[http://parts.igem.org/Part:BBa_K1419004 BBa_K1419004]	Device	TEV Protease	Harland Brandon	729DFDGFHFDHF
[http://parts.igem.org/Part:BBa_K1419005 BBa_K1419005]	Device	Lamp2B-Clover	Harland Brandon	2241	Yes

Expression and localization of RVG-Lamp2B protein construct

To test if our pcDNA3.0-RVG-Lamp2B construct was being appropriately expressed and folded, we fused Clover (a green fluorescent protein) to the C-terminus of Lamp2B. Using the Phyre2 server, we were able to generate a model of the predicted structure of our RVG-Lamp2B-Clover fusion protein based on homology and multi-template ab initio modeling.

Figure 1: The predicted structure of the RVG-Lamp2B-Clover fusion protein. The Clover domain is obvious in red with Lamp2B below in green and the RVG domain evident at the bottom in blue.

The pcDNA3.0-RVG-Lamp2B-Clover plasmid and a pcDNA3.0-Clover control were lipofected into separate Human Embryonic Kidney (HEK-293) cell cultures (HEK-293 cells are a resilient and rapidly dividing cell line also known to produce exosomes). In both lipofected cultures, there was elevated cytosolic green fluorescence relative to non-lipofected controls, which confirms expression and proper folding of the protein construct.

To confirm that the RVG-Lamp2B-Clover protein construct was being targeted to exosomal membranes (and were not simply cytosolic), culture media was collected from the lipofected and control HEK-293 cell cultures and exosomes were purified via ultracentrifugation. After lysis, the exosomes isolated from pcDNA3.0-RVG-Lamp2B-Clover lipofected cultures demonstrated an elevated level of fluorescence (505-620nm) that corresponds to the emission wavelength of Clover (515nm) indicating appropriate targeting of the engineered protein to exosomal membranes.

Figure 2: Lysed exosomes isolated from pcDNA3.0-RVG-Lamp2B-Clover lipofected HEK-293 cells demonstrate elevated fluorescence in the 505-620nm range relative to control and unlysed samples.</p>

<p><b>Figure 3: Confocal Image of isolated exosomes carrying the Lamp2B-clover construct. Description goes here.

Figure 1. Confocal Image of HEK cells transfected with Lamp2B-clover construct. Description goes here.

Figure 1. Confocal Image of HEK cells transfected with Lamp2B-clover construct. Description goes here.