Name: PowerPoint Presentation
Uploaded: 2022-02-03
Duration: 5 min
Description: Designing , construction and characterization of genetically encoded FRET based nanosensor for in vivo measurement of Exosomes Reshma Bano , Mohd . Mohsin * Metabolic Engineering Lab, Department of Biosciences Jamia Millia Islamia , New Delhi-110025.

Designing , construction and characterization of genetically encoded FRET based nanosensor for in vivo measurement of Exosomes Reshma Bano , Mohd . Mohsin * Metabolic Engineering Lab, Department of Biosciences Jamia Millia Islamia , New Delhi-110025. 

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Designing , construction and characterization of genetically encoded FRET based  nanosensor  for in vivo measurement of  Exosomes Reshma   Bano ,  Mohd .  Mohsin * Metabolic Engineering Lab, Department of Biosciences Jamia   Millia   Islamia , New Delhi-110025

The genetically encoded  nanosensors  uses Fluorescence Resonance Energy Transfer (FRET) for optical detection of molecular events within the cell. Small molecule sensors expressed from DNA vectors are powerful tools that help elucidate various events and mechanisms, thus providing knowledge of the biological systems. The engineered FRET based  nanosensor  uses MIG-6 gene as  exosomal  EGFR-binding domain sandwiched between a donor  fluorophore , ECFP and an acceptor  fluorophore , Venus for monitoring the  exosomes . Exosomes  perform the role of mediators of intercellular communication by conveying biological information between cells. MIG-6 is a binding partner of  egfr , which is overexpressed in cancer derived  exosomes . The  MIG6 (mitogen-induced gene 6) interacts with and inhibits the kinase domains of EGFR.   The atomic coordinates are deposited at the Protein Data Bank under accession codes 2RF9, 2RFD and 2RFE.

Binding proteins (MIG-6) can be exploited to make genetically encoded FRET sensors. Genetically encoded FRET sensors allow real-time, non-invasive  exosomes  measurements. FRET –based sensors can be used for detection of the  exosomes  early diagnosis in various disease conditions.

The engineered sensor proteins can be used for monitoring level of  exosomal  biomarkers which would further help in understanding cancerous or other disease condition within the cell .

The First author RB is thankful of MANF, UGC-  Govt  of India for providing the fund in the form of Senior Research fellowship.

DIAGRAMMATIC REPRESENTATION FRET-BASED NANOSENSOR FOR EXOSOMES

Designing & construction of FRET-based genetically encoded  nanosensor  of  exosomes . Expression and purification of  nanosensor . In vitro  and  In vivo  characterization of the  nanosensors . Monitoring the  exosomes  levels under  in vivo  conditions in bacteria, and animal cell by using  nanosensors .

We have successfully cloned MIG 6 genes between ECFP (donor  fluorophore ) and Venus (acceptor  fluorophore ) at N and C terminal respectively in order to construct the  nanosensor  in  pRSET -B vector.

Amplification of  pRSET -B (A) and clone of MIG6 in  pRSET -B_ECFP_VENUS backbone (B )

Designing of the  Nanosensor  after gene synthesis  MIG6 by  GeneScript .  Bacterial cloning expression vector  pRSET -B was used for the cloning and the expression of the sensor protein. MIG 6 genes cloned between ECFP and Venus in order to construct the  nanosensor . The chimeric construct was transformed in E. coli  rossetta  strain for expression of  nanosensor  proteins. Exosomes  are isolated from  A549   cell line to characterized the  nanosensor  through  qEV  column.   In   vitro  characterization of  nanosensor  was done on synergy  microplate  reader. Ratiometric  analysis of  exosome’s  sensor was  carried out using confocal microscopy.

The chimeric fusion protein carries N-terminal His6 affinity tag for purification. The protein was purified using Ni- NTA affinity chromatography. Purity of the chimeric proteins were verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

In Vivo  Expression of  exosome’s  sensor in Bacterial Cells (Confocal microscopy)

The addition of  exosomes  with the  nanosensor’s  protein, ECFP and Venus's emission spectral profile indicate relevant variation in the emission intensities The emission intensity of ECFP decreases and the emission intensity of Venus increase subsequently .

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