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Part:BBa_K4829003:Experience

Designed by: Aditya Kamath Ammembal, Dr. Raghavan Varadarajan, Dr. Debajyoti Chakraborty   Group: iGEM23_IISc-Bengaluru   (2023-10-04)


Applications of BBa_K4829003

This part, on In vitro Transcription followed by mRNA purification gives us the mRNA strand coding for the BBa K4829002. This mRNA, when transfected into HeLa cells (We used 2 micrograms of mRNA with 2 microlitres of lipofectamine MessengerMax), gives protein in the supernatant after a mere 6 hours post-transfection. This was detected by a western blot:

  • hela-blot.png 
  Figure 1. Production of the Protein at the 31kDa mark 6hrs post-transfection.

The mRNA synthesised from this biobrick can further be used to make LNP's loaded with the mRNA, using a microfluidic device, specified in our wiki. The characterisation of the LNP's has been done using DLS. We have measured the radius as well as the Zeta potential of the LNP's, which are satisfactory. The data is encapsulated by the following images:

  • zeta-potent-graph.jpg
 Figure 2.  The Graph of the Zeta potential as measured by the ZetasizerNano by Malvern Panalytical
  • radius-data.png
 Figure 3. The radius of the LNps as measured by the ZetasizerNano by Malvern Panalytical ]]


The efficiency of the encapsulation of the mRNA was measured using the Ribogreen assay, with the undialysed sample of LNPs.

  • ribogreen-vf.png
 Figure 4. Graph of the curve fit between fluorescence and the RNA that is unencapsulated

Please note that though we have not shown the calculation of how to calculate encapsulation efficiency here, the same can be found on the results page of iGEM IISc Bengaluru.

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