Part:BBa_K2412001
shRNA circuit for eGFP knockdown
This is a IPTG-inducible circuit with a T7 promoter that produces an shRNA that knocks down commercialized eGFP.
Usage and Biology
This circuit produces shRNA that is designed to knock down the expression of eGFP. The IPTG-inducible T7 promoter and the T7 enhancer is a very strong promoter system, so high production of this shRNA occurs when the cells are IPTG induced. We used this circuit to produce shRNA in BL21 (DE3) E. coli cells. When delivered to the cytoplasm of cells, this shRNA knocks down expression of its target gene, commercial eGFP, through RNA interference. In RNA interference, the shRNA is first processed by the enzyme Dicer to form siRNA, and the antisense of this siRNA associates with other proteins and the mRNA of eGFP to form the RISC complex. The formation of the RISC complex causes the mRNA of the eGFP to be cleaved, effectively inhibiting production of eGFP for the cleaved mRNA.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 43
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
We produced the shRNA in vitro using the New England Biolabs T7 expression kit to use in the lipofectamine assay. After the in vitro shRNA is successful, we reran the lipofectamine assay with shRNA that was produced in vivo using IPTG induction. The shRNA produced in vivo was isolated using the miRNeasy kit. We extracted a significant quantity of shRNA using both in vitro expression and in vivo expression. In in vitro expression, the concentration of shRNA was about 2230 ng/uL. In in vivo expression, concentrations ranged from 850 ng/uL to 1070 ng/uL. The lipofectamine assay would test whether the shRNA successfully knocks down the eGFP in mammalian cells. In this assay, the shRNA would be introduced into liposomes, and then the liposomes would be introduced into the mammalian cell line. The liposomes would fuse with the cell membrane of the mammalian cells and then the shRNA would enter the cells. Upon entry, the shRNA would knock down the expression of eGFP. The effectiveness of the knockdown of eGFP from the shRNA is measured using a flow cytometer. The flow cytometer measures the GFP florescence in every cell and gives a statistical distribution of the florescence. The results from the synthesized shRNA is compared to a negative control, where none of the shRNA is introduced to the mammalian cells during the lipofectamine assay. The results will also be compared to a positive control, where we used siRNA designed and proven to knock down eGFP from Thermo Fisher. We analyze the decrease in florescence to determine the effectiveness of the shRNA-mediated knockdown of GFP.
This graph illustrates the data collected from the flow cytometer after our lipofectamine assay.
Figure 1: The x-axis shows the negative control (PBS), the positive controls (siRNA), and the experimental shRNA designed to inhibit eGFP (shRNA). The y-axis shows the percentage of mammalian cells that were fluorescing eGFP (defined as eGFP positive). The error bars are 2 standard error, or a 95% confidence interval. As can be seen, the confidence intervals for the negative control and the experimental shRNA do not overlap, which shows that this difference is statistically significant. This indicates that our shRNA successfully knocks down eGFP expression in HeLa cells.
We have confirmed the sequencing of this BioBrick, and the sequencing data is below.
| None |