Part:BBa_K2623032

A composite part linked by BBa_K2623024, BBa_K2623026 and BBa_K2623027

Summary

Our OMVs module is designed for siRNA delivery and then for cancer treatment. To demonstrate the conjugation of the L7Ae-C/D_box inside OMVs, we linked ST/SC part with the box part (BBa_K2623027) to form our total circuit (BBa_K2623032).The box part can code siRNA target for Kras in human pancreatic ductal adenocarcinoma (PDAC) with a C/Dbox RNA structure to bind with L7Ae.

Usage and Biology

Under the induction of IPTG and Arabinose, this part can code SpyTag-OmpA, SpyCatcher-L7Ae and siRNA-C/D_box to be encapsulated into OMVs through the isopeptide bond formed between SpyTag/SpyCatcher and the linkage between L7Ae and C/D_box (Figure 1).

Figure 1. Schematic illustration of our OMVs.

Characterization

We isolated OMVs according to our protocols and then stained the OMVs isolates with SYTO^TMRNASelect^TM at 10 μM . The stained isolates were analyzed by HSFCM (Figure 2).

Figure 2. HSFCM analysis of OMVs stained with SYTOTM RNASelectTM.

Figure 2 are bivariate dot-plots of green fluorescence versus side scattering (SSC) for our OMVs isolates. E. coli BL21 transfected with BBa_K2623029 and BBa_K2623032 respectively were cultured in 100 mL OMVs-free LB culture for about 5 hours to get a OD600 at 0.6-0.8, in which BBa_K2623029 was set as negative control. Then Arabinose was added to a final concentration at 0.2% and after incubation for another 2 hours, IPTG was added to a final concentration at 0.5 mM and then nurtured the bacteria overnight. OMVs were isolated according to our protocols and stained with SYTO^TM RNASelect^TM. The stain OMVs were analyzed by HSFCM. As we expected, OMVs isolated from BBa_K2623032 could secret more OMVs containing with RNA stained positively by SYTO^TM RNASelect^TM than BBa_K2623029, indicating that our L7Ae and C/D_box could fulfill their function. It’s a pity that we didn’t have cell culture experiment to test our siOMVs to silence Kras in human pancreatic ductal adenocarcinoma (PDAC). In our future plans, we’ll finish our cell experiment and demonstrate the function of our siOMVs.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal NheI site found at 1965
  Illegal NheI site found at 1988
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 3974
  Illegal BamHI site found at 3850
  Illegal BamHI site found at 4004
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 65
  Illegal AgeI site found at 3685
  Illegal AgeI site found at 4010
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 3667