Part:BBa_K5353065

1108 LentiV-Lamp2b/hTRPC1/HA: Neo

After purify the plasmid DNA through midi-prep, we utilize them to package the lentivirus together with DVPR and VSV-G. We utilize these lentivirus to infect HEK293T cell and the successfully infected cells will express the fusion protion of Lamp2b-hTRPC1 with HA-Tag. The Lamp2b sequence is for the transportation of hTRPC1 to the exosome. hTRPC1 is a cation channel that is permeable to calcium (Ca2+) will be used for the calcium overload. The lentivirus gene sequence could be integrated into about HEK293T cell genome by utilizing G418 selection (by the Neo gene).

The plasmid information.

The designed DNA was constructed and we used the midi-prep preparation kit to conduct the plasmid isolation and purification of the plasmid DNA (Table 1). The inserts in each construct were further validated by the PCR reaction (Figure 1. and Figure 2).

Table 1.DNA concentrations extracted and purified.

Figure 1. Gel electrophoresis results 1.

Figure 2. Gel electrophoresis results 2.

The size of the exosomes is extremely small, approximately 30–140 nm, membrane-enclosed molecules. The halo-like structure is the exosomes (Figure 3.1 and Figure 3.2), and the light dots shown on the background are salts. The normal and well-functioning exosomes are standard round, while the broken and unhealthy exosomes with an elliptical collapse structure were also observed (Figure 3.3).

Figure 3.1 Exosomes with normal structures.

Figure 3.2 Exosomes tangled together.

Figure 3.3 Exosomes with elliptical collapse structure.

We used DLS to provide valuable insights into the size distribution and polydispersity of exosomes, which typically range in size from 200 to 300 nanometers.(Figure 4.)

Figure 4. Exosome size distribution of sample 1108-1.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 843
  Illegal XbaI site found at 1593
  Illegal PstI site found at 715
  Illegal PstI site found at 1101
  Illegal PstI site found at 1465
  Illegal PstI site found at 1851
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 715
  Illegal PstI site found at 1101
  Illegal PstI site found at 1465
  Illegal PstI site found at 1851
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 4179
  Illegal BamHI site found at 2574
  Illegal XhoI site found at 3869
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 843
  Illegal XbaI site found at 1593
  Illegal PstI site found at 715
  Illegal PstI site found at 1101
  Illegal PstI site found at 1465
  Illegal PstI site found at 1851
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 843
  Illegal XbaI site found at 1593
  Illegal PstI site found at 715
  Illegal PstI site found at 1101
  Illegal PstI site found at 1465
  Illegal PstI site found at 1851
  
• 1000
  COMPATIBLE WITH RFC[1000]