Difference between revisions of "Part:BBa K2660001"

Revision as of 02:37, 18 October 2018

T7 promoter driving 6-his tagged GFP with penetratin

GFP with his tag for purification under strong promoter T7. This part has the cell-penetrating peptide (CPP) penetratin (BBa_K2660000) and was used by iGEM Unesp Brazil 2018 Team to evaluate this CPP in Caco-2 cell culture. We used E.coli BL21 (DE3) for expression of this part: addition of 1mM IPTG leads to expression of the T7 DNA polymerase which in turn drives expression of GFP.

Experiments
We performed an in vitro permeability test in caco-2 cell monolayer. Caco-2 cells are human colon carcinoma cells that slowly differentiate into a monolayer of intestinal epithelial cells when cultured in a permeable support (HUBATSCH et al., 2007). We cultured caco-2 cells on a porous plate filter in a transwell system (Figure 1). After 21 days of growth, cell differentiation was observed forming a monolayer of caco-2 cells.

Figure 1. In vitro permeability test in caco-2 cell monolayer. Caco-2 cells were cultured in DMEM medium in a transwell plate for differentiation and further permeability test. GFP (control) and Penetratin-GFP were added to the apical compartment of the transwell plate.

At the 21st day, we added about the same amount of GFP (control) or Penetratin-GFP to the apical compartment of the transwell plate (3 wells for each treatment). The plate was incubated for further 4h. In order to determine whether the cell layer was viable, we measured the trans-epithelial electrical resistance (TERR). Inserts presenting EQ values at least 260 ± 65 Ohm x cm2 are considered ideal for testing (HUBATSCH et al., 2007). Our monolayers presented EQ values between 253 and 350 Ohm x cm². Samples from apical and basal compartments were taken for fluorescence analysis at beginning and after 4h of assay. Only the GFP fused to penetratin was able to cross the differentiated caco-2 monolayer. From the total penetratin-GFP added to the apical compartment at time 0, about 71% crossed the monolayer after 4h.

Figure 2. GFP fluorescence measured in the apical and basal compartments of the transwell plate. Fluorescence found in the basal compartment indicates that penetratin promoted permeation of GFP through the caco-2 monolayer.

References Hubatsch, I. et al. Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers. Nature Protocols 2(9), 2111-2119, 2007.

Sequence and Features