Part:BBa_K4737020
prpsM::T7 RNA Polymerase-T7 Promoter::GFP-shRNA
To express T7 RNA polymerase and GFP-shRNA constitutively.
Usage and Biology
We selected human gastric adenocarcinoma cells BGC823 and transiently transfected with the GFP eukaryotic expression vector. After the fluorescent protein was effectively expressed in BGC-823 24h after transfection, the cells were co-cultured with the engineered bacteria expressing shRNA-GFP. We chose the multiplicity of infection (MOI) of cells and engineered bacteria (VNP20009-shGFP) as 1:500, at the same time, we also used VNP20009 without shRNA-GFP at an MOI of 1:500 to infect tumor cells as negative control results. We observed the results under fluorescence microscope after 24 hours (Figure. 4). Compared with the VNP20009 control, the tumor cells’ fluorescence was significantly weakened after VNP20009-shGFPinfection, indicating that the engineered bacteria actually delivered shRNA-GFP after entering the tumor cells, and effectively down-regulated the gene expression.
Figure 1. Results of shGFP delivery by bacterial infection 24 hours later. Both the engineered bacteria and VNP20009 infections were at the MOI of 1:500. Control was BGC-823 not transfected with the GFP transient plasmid.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 1
Illegal PstI site found at 34 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 34
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 1
Illegal PstI site found at 34 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 1
Illegal PstI site found at 34 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1985
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