Team:Munich/Composite Part

Alive

COMPOSITE PARTS

iGEM Munich Composite Part List

NameTypeDescriptionDesignerLength
   BBa_K3113200RegulatorypCAGTheresa Keil1712
  BBa_K3113201CodingpCAG_XPATheresa Keil2702
   BBa_K3113316ProjectpCAG_iRFPAlejandro Salinas Illarena, Joshua Hesse2969
   BBa_K3113202ProjectpCAG_fluc_MS2Alejandro Salinas Illarena3736
   BBa_K3113203ProjectpCAG_fluc_C/DboxAlejandro Salinas Illarena, Joshua Hesse3746
   BBa_K3113306ProjectpCAG_P10SN-L7AeAlejandro Salinas Illarena2492
   BBa_K3113307ProjectpCAG_P10SN-Flag-L7AeAlejandro Salinas Illarena2537
   BBa_K3113308ProjectpCAG_P10SN-MCPAlejandro Salinas Illarena2513
   BBa_K3113309ProjectpCAG_P10SN-mEos4bAlejandro Salinas Illarena2816
   BBa_K3113310ProjectpCAG_DHD154b-Flag-L7AeAlejandro Salinas Illarena2678
   BBa_K3113311ProjectpCAG_DHD154b-MCPAlejandro Salinas Illarena2663
   BBa_K3113312ProjectpCAG_DHD154b-mEos4bAlejandro Salinas Illarena2957
   BBa_K3113321ProjectpCAG_flucAlejandro Salinas Illarena3671
   BBa_K3113322ProjectpCAG_nluc_C/DboxAlejandro Salinas Illarena2609
NameTypeDescriptionDesignerLength
   BBa_K3113204ProjectpCAG_CD63_HiBit_L7AeJoshua Hesse3188
   BBa_K3113205ProjectpCAG_CD63_HiBit_MCPJoshua Hesse3185
   BBa_K3113206ProjectpCAG_Lamp2bJoshua Hesse3389
   BBa_K3113207ProjectpCAG_CD63_Ser161_6xHis_HiBit_L7AeSarah Brajkovic, Joshua Hesse3218
   BBa_K3113208ProjectpCAG_CD63_Ser161_6xHis_HiBit_MCPSarah Brajkovic, Joshua Hesse3215
   BBa_K3113209ProjectpCAG_CD63_Ser161_6xHis_HiBit_P9SNSarah Brajkovic, Joshua Hesse2939
   BBa_K3113210ProjectpCAG_CD63_Asn180_6xHis_Hibit_L7AeSarah Brajkovic, Joshua Hesse3218
   BBa_K3113211ProjectpCAG_CD63_Asn180_6xHis_HiBit_MCPSarah Brajkovic, Joshua Hesse3221
   BBa_K3113212ProjectpCAG_CD63_Ser161_6xHis_Hibit_DHD154aSarah Brajkovic, Joshua Hesse3065
NameTypeDescriptionDesignerLength
   BBa_K3113313ProjectpCAG_hArc-HiBitAlejandro Salinas Illarena3278
   BBa_K3113314ProjectpCAG_Avilabel-PSGL1-TMandCTAlejandro Salinas Illarena4106
   BBa_K3113315ProjectpCAG_His6-PSGL1-TMandCTAlejandro Salinas Illarena2423
   BBa_K3113318ProjectpCAG_mGag-HiBit-L7AeAlejandro Salinas Illarena3950
   BBa_K3113319ProjectpCAG_mGag-HiBit-CC9Alejandro Salinas Illarena3689
   BBa_K3113320ProjectpCAG_mGag-HiBit-DHD154aAlejandro Salinas Illarena3815



Best New Composite Parts

Our best new composite part is also the part we chose for the validation: pCAG_Gag-HiBit-L7Ae, BBa_K3113302. This construct forms virus-like particles (VLPs) which can be detected via a split luciferase assay. Through the RNA binding protein, the vesicles can be loaded with RNA.

Transmission Electron Microscopy (TEM)


To validate that our vesicles are intact and properly shaped, we prepared purified VLPs and exosomes for transmission electron microscopy (TEM). This microscopy technique is based on a high-energy beam of electrons shining through a very thin sample fixed on a grid and allows high-resolution imaging.


Size distribution frequency of VLPs in HEK293T
Figure 1: Analysis of particle diameter distribution for VLPs secreted from HEK293T cells. Results have been calculated from 50 (unloaded, red) and 116 (loaded, blue) particles, respectively. A Gaussian fit was performed on the data and the average diameter was calculated as approximately 73 nm and 96 nm for unloaded and loaded VLPs, respectively.


Dynamic Light Scattering (DLS)


To further characterize our vesicles and determine the size distribution and sample homogeneity, we performed Dynamic Light Scattering (DLS) to determine the size and shape of our vesicles.
DLS measurements of Virus-like particles showed a narrow Gaussian size distribution indicating that the samples are very homogenous. Interestingly, a shift of about 30 nm is seen between cargo-loaded and unloaded VLPs; cargo-loaded particles have a mean diameter of 104 ± 14 nm, whereas unloaded vesicles showed a mean diameter of 71 ± 11 nm.


Size distribution frequency of VLPs in HEK293T
Figure 2: Dynamic light scattering (DLS) measurement of VLPs. Purified VLPs either with or without the adapter protein L7Ae, were measured. Unloaded and loaded vesicles showed a narrow size distribution with mean diameters of approximately 71 and 104 nm, respectively.


Split-luciferase bioluminescent assay: The HiBiT Assay


To prove that the BioBrick Part we designed works as expected, we performed a HiBit split-luciferase assay, which shows luminescent signal detected in fully formed VLPs. On the graph below, the data shows that engineered Gag protein has been expressed in HEK293T cells. Further on, based on this data, we have calculated the export to be in approx. 50% for transfection with as well as without adapter construct. Conclusivelly, we report Gag has successfully assembled into VLPs.


A generic square placeholder image with rounded corners in a figure.
Figure 3: Secretion efficiency of Gag vesicles calculated from HiBiT-measurements. Vesicles containing the adapter L7Ae as well as empty vesicles lacking the RNA binding protein (RBP) were secreted from HEK293T cells with comparable efficiency. In contrast, control samples transfected with mock DNA did not show any secretion. Cell signal shown in absolute values corresponds to extrapolated cellular content data. Absolute supernatant values are calculated by subtracting extrapolated values from unlysed supernatant from corresponding lysed supernatant values. Measurements were performed for n = 6 biological replicates in a 96-well format.


Affinity Purification


We have established Heparin affinity chromatography as our method of choice for VLP purification. Figure 4 shows an exemplary chromatogram of a purification run with an increasing NaCl gradient for elution. Fractions of 1 ml were collected for each NaCl concentration up to 2 M and analyzed with an HiBiT assay to determine the absolute amount of Gag. After elution, we collected another 10 fractions with 2 M NaCl. As it can be seen, these fractions showed almost no HiBiT signal, indicating that all vesicles were eluted from the column. In total, approximately 4 % of the HiBiT signal was detected in the flow-through, 5% in the wash and 55% in the elution fraction.


A generic square placeholder image with rounded corners in a figure.
Figure 4:Heparin affinity purification of Virus-Like-Particles (VLPs). We analysed the individual fractions using the HiBiT assay. Approximately 40 % of the HiBiT signal was detected in the flow-through, 5 % in the wash and 55 % in the elution fraction.


qPCR Analysis


Finally, the BioBrick Part we designed works as expected since FLuc mRNA is successfully transported into VLPs through L7Ae-C/D box interaction. This has been proven through qPCR analysis of VLP content. FLuc mRNA cellular content is calculated with delta Ct analysis, where GAPDH housekeeping gene is used for normalization. Alternatively, this can also be calculated with a standard curve method. Also taking cell confluency (70-80%) into account, we can calculate the accurate amount of Fluc RNA content/cell. On the graph below you can observe a 100-fold increase in FLuc signal/cell with our validated VLP BioBrick Part, compared to control. Furthermore, this part performs 20-fold better than a parallel construct used for exosomes.


A generic square placeholder image with rounded corners in a figure.
Figure 6: FLuc mRNA detection in cells producing exosomes (blue) and VLPs (red). All cells transfected with FLuc mRNA show detectable expression levels inside cells, which was significantly higher than for untransfected mock controls. Measurements were performed in technical duplicates (n = 2) with EEJ primers. Quantification was done via ͍delta Ct method.

Figure 7: Cargo mRNA export in exosomes and VLPs quantified via qPCR.
Figure 7: Cargo mRNA export in exosomes and VLPs quantified via qPCR. Both exosomes (blue) and VLPs (red) containing the RNA adapter L7Ae were capable of exporting FLuc mRNA whereas control cells lacking the RNA binding protein (RBP) showed no significant export. Measurements were performed in technical duplicates (n = 2) with EEJ primers. Quantification was done via standard curve measurements and assuming a confluent well with 350.000 HEK293T cells/cm2 at the time of harvesting.