Team:Munich/Basic Part

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BASIC PARTS

iGEM Munich Basic Part List

NameTypeDescriptionDesignerLength
   BBa_K3113000RegulatoryCAG promotor Theresa Keil1671
   BBa_K3113002T7T7 promotorTheresa Keil23
  BBa_K3113003RBSkozakTheresa Keil9
   BBa_K3113004TerminatorpolyATheresa Keil272
  BBa_K3113005CodingFLucTheresa Keil1650
  BBa_K3113006RNAMS2-StemloopTheresa Keil18
  BBa_K3113007RNAC/D-BoxTheresa Keil29
  BBa_K3113008CodingHiBiTTheresa Keil33
  BBa_K3113009CodingL7AeTheresa Keil357
  BBa_K3113010CodingMCPTheresa Keil387
  BBa_K3113011Tag6xHisTheresa Keil18
  BBa_K3113012CodingXPATheresa Keil681
  BBa_K3113014CodingNLucTheresa Keil513
   BBa_K3113068CodingDHD154aAlejandro Salinas Illarena216
   BBa_K3113069CodingDHD154bAlejandro Salinas Illarena231
  BBa_K3113102CodingP9SNTheresa Keil96
  BBa_K3113103CodingP10SNTheresa Keil99
  BBa_K3113104CodingP11SNTheresa Keil96
  BBa_K3113111CodingFlag-TagTheresa Keil24
  BBa_K3113115CodingmEos4bTheresa Keil678
  BBa_K3113118CodingiRFPTheresa Keil948
NameTypeDescriptionDesignerLength
    BBa_K3113051CodingCD63_Ser161_6xHisSarah Brajkovic, Joshua Hesse744
    BBa_K3113072CodingCD63_Asn180_6xHisSarah Brajkovic744
  BBa_K3113050CodingCD63Theresa Keil714
   BBa_K3113052Protein_DomainLamp2b-1Joshua Hesse108
   BBa_K3113053Protein_DomainLamp2b-2Joshua Hesse1242
NameTypeDescriptionDesignerLength
    BBa_K3113302ProjectpCAG_Gag-HiBit-L7AeAlejandro Salinas Illarena3950
   BBa_K3113100CodingGag HIV-1Alejandro Salinas Illarena1500
   BBa_K3113101CodingmGagAlejandro Salinas Illarena1500
   BBa_K3113106CodingHis6-PSGL1-TMandCTAlejandro Salinas Illarena402
  BBa_K3113107CodingAvilabel-PSGL1-TMandCTTheresa Keil2085
   BBa_K3113116CodinghArcAlejandro Salinas Illarena1188
NameTypeDescriptionDesignerLength
   BBa_K3113001ScarLinker CAG_T7Theresa Keil9
   BBa_K3113013ScarLinker_XPA-polyATheresa Keil37
   BBa_K3113054ScarLinker_Fluc-MS2Theresa Keil43
   BBa_K3113055ScarLinker_(3´)Fluc_MS2-polyATheresa Keil41
   BBa_K3113056ScarLinker_Fluc-C/D-BoxTheresa Keil43
   BBa_K3113057ScarLinker_Fluc_C/D-Box-polyAAlejandro Salinas Illarena, Joshua Hesse40
   BBa_K3113058ScarLinker_CD63-Hibit_MCPTheresa Keil21
   BBa_K3113059ScarLinker_CD63-HiBit_L7AeAlejandro Salinas Illarena, Joshua Hesse30
   BBa_K3113060ScarLinker_CD63_His-HiBit_MCPAlejandro Salinas Illarena, Joshua Hesse26
   BBa_K3113061ScarLinker_CD63_His-HiBit_L7AeAlejandro Salinas Illarena, Joshua Hesse35
   BBa_K3113062ScarLinker_HiBit-MCPTheresa Keil9
   BBa_K3113063ScarLinker_HiBit-L7AeTheresa Keil33
   BBa_K3113064ScarLinker_MCP/L7Ae/iRFP-polyAJoshua Hesse37
   BBa_K3113065ScarLinker_Lamp2b2-polyAJoshua Hesse37
  BBa_K3113066ScarLinker_CD63_His-HiBit-CC9Theresa Keil15
  BBa_K3113067ScarLinker_HiBit_DHD154aTheresa Keil21
  BBa_K3113070ScarLinker_DHD154b-FlagTheresa Keil27
  BBa_K3113071ScarLinker_DHD154b-MCPTheresa Keil24
  BBa_K3113073ScarLinker_CD63_Asn180_6xHis-HiBitTheresa Keil27
  BBa_K3113108ScarLinker_Gag-HiBitTheresa Keil24
  BBa_K3113109ScarLinker_HiBit-polyATheresa Keil58
   BBa_K3113110ScarLinker_HiBit-P9SN/P11SNAlejandro Salinas Illarena15
  BBa_K3113112ScarLinker_P10SN-FlagTheresa Keil18
  BBa_K3113113ScarLinker_Flag-L7AeTheresa Keil18
  BBa_K3113114ScarLinker_VLP-polyATheresa Keil37
  BBa_K3113117ScarLinker_hArc-HiBitTheresa Keil21


Best New Basic Part

Our best basic part is the CD63-6xHis construct (BBa_K3113051). To facilitate the purification of intact exosomes, we engineered the exosomal marker CD63 by integrating a polyhistidine repeat into the large extracellular loop of the tetraspanin. Based on SWISS-MODEL, a protein structure homology-modelling server, we were able to model CD63 and to search for sites within CD63 that are accessible.




Figure 1: CD63 structure based on CD81. Position of incorporated 6xHis-tag is highlighted in red.

Usage

This part was designed to allow the purification of exosomes via affinity chromatography. The exosomal marker protein CD63 belongs to the family of tetraspanins and is therefore composed of four alpha-helical transmembrane domains with two extracellular loops. Both the N- and the C-terminus point towards the inside of exosomes, rendering terminal His-tagging useless for affinity purification of exosomes. Therefore, we innovatively fused a 6xHis-tag to an external loop of the exosomal marker CD63. Specifically, the six histidines were added after position Ser161 based on a structural model for CD63 generated with swissmodel.expasy.org and based on the structure of the related tetraspanin CD81. To our knowledge, Ni-NTA affinity chromatography has not been previously been used to purify exosomes, it has only been applied to other His-tagged membrane structures.(Alves, N.J.et al 2017) BBa_K3113051 is thus an improvement from iGEM 2018 XJTLU-China's part BBa_K2619003, which just contains the human CD63 sequence.

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Figure 2:CD63 with a polyhistidine integrated in the large extracellular loop.


Biology

CD63 is a Tetraspanin. Tetraspanins are a superfamily of cell surface-associated membrane proteins with four transmembrane domains. CD63 was the first characterized Tetraspanin. It is abundantly present in late endosomes and lysosomes as well as exosomes. The gene is located on the human chromosome 12q13. Although the intracellular function of CD63 remains to be established, a number of studies performed in different cell types implicate a role for CD63 in intracellular transport of other proteins.



Characterisation


Purification

We performed a Ni-NTA affinity chromatography to purify our modified exosomes from the supernatant of HEK293T and MIN6-K8 cells. Afterwards, a HiBiT assay was performed with the Flow-through, wash and elution phases to measure the content of CD63-Ser161-6xHis which was fused to a HiBit split luciferase. Both parts were put under the control of the mammalian constitutive promoter CAG (BBa_K3113000), contained the same kozak sequence (BBa_K3113003) before the CD63 ORF, and had the same polyadenylation signal motive (BBa_K3113004) at the end. When applying exosomes containing either the wild-type CD63 or our internally His-tagged CD63 on Ni-NTA columns, only the latter get purified efficiently (Figure 1). Thus, purification of exosomes does not happen due to unspecific interaction with the column material but is enabled by the His-tag on the extracellular loop. Moreover, the vesicle quality along the purification can be monitored with the HiBiT-tag inside the exosomes. Since the HiBiT signal of free His-CD63 outside exosomes is about 20 times lower than the total His-CD63 in the elution fraction (Figure 2), it can be concluded that most of it is still enclosed in exosomes. Therefore whole exosomes are eluted from the column using our improved BBa_K3113051 .

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Figure 2: Ni-NTA affinity purification of exosomes monitored via the HiBiT-tag on CD63. Tested were exosomes containing wild-type CD63 and His-tagged CD63 between positions Ser161 and K162. Data from three independent purification experiments.


HiBiT

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Figure 3: Quality check of loaded and eluted exosomes during the Ni-NTA affinity purification monitored via the HiBiT-tag on His-CD63.

Moreover, the vesicle quality along the purification can be monitored with the HiBiT-tag inside the exosomes. Since the HiBiT signal of free His-CD63 outside exosomes is about 20 times lower than the total His-CD63 in the elution fraction (Figure 2), it can be concluded that most of it is still enclosed in exosomes. Therefore whole exosomes are eluted from the column using our improved BBa_K3113051.



Transmission Electron Microscopy

Histogramm of HEK: exosomes.
Figure 4: Characterization of vesicles was performed by Transmission Electron Microscopy, TEM (A+B). Grids with exosomes (A) were negatively stained with 1 % uranyl acetate for TEM. The diameter for VLPs and exosomes was precisely determined with DLS. It was revealed that the exosomes differ in size depending on the secreted organism.
A difference in size exosomes from MIN6-K8- and HEK293T-cells can be observed, but it is not statistical significant.


Western Blot

Histogramm of HEK: exosomes.
Figure 5: The presence of vesicular components modularly composed with coiled-coils and directly fused could be proven by western blotting. Top left) the exosomal marker cdc63 can be visualized with primary anti-cdc63 mouse-antibody and secondary anti-mouse antibody - horse radish peroxidase (HRP) fusion. CDC63 does not run as a tight band on the blot because of glycosylation patterns and its nature as a membrane protein. Top right) Gag-protein is determined at around 70 kDa. The fusion construct Gag-HiBiT-L7Ae shows some degradation corresponding to the molecular weight of L7Ae cleavage. Bottom) MCP RNA-binding proteins can be shown with anti-MCP antibodies.

To confirm the HiBiT data of the purification, a Western Blot was performed with the elution phases of the CD63-6xHis constructs as well as the concentrated supernatant of exosomes with endogenous CD63.