Difference between revisions of "Team:HZAU-China/Improve"
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| − | <p>Microscopy shows that E. coli expressed Lpp-OmpA-RGD induced by 0.1mM IPTG can bind to αVβ3-positive | + | <p>Microscopy shows that <sub>E. coli</sub> expressed Lpp-OmpA-RGD induced by 0.1mM IPTG can bind to αVβ3-positive |
MDA-MB-231 cell line. Red arrow points the location of bacteria (Figure 2). But cannot bind to | MDA-MB-231 cell line. Red arrow points the location of bacteria (Figure 2). But cannot bind to | ||
αVβ3-negative MCF7 cell line (Figure 3). We also use BBa_J36850 as a control. This strain cannot | αVβ3-negative MCF7 cell line (Figure 3). We also use BBa_J36850 as a control. This strain cannot | ||
Revision as of 13:47, 17 October 2018
We gave the surface display system from BBa_J36850 a new function through displaying a RGD motif on the Lpp-OmpA. RGD motif can specifically bind to αVβ3, a biomarker of cancer cells1. We determine the surface display site on the third loop of Lpp-OmpA through homology modelling (Figure 1). This part is under the control of lac promoter.
Figure 1. Homology modelling result of Lpp-OmpA-RGD. Red arrow shows the location of RGD motif.
Microscopy shows that E. coli expressed Lpp-OmpA-RGD induced by 0.1mM IPTG can bind to αVβ3-positive MDA-MB-231 cell line. Red arrow points the location of bacteria (Figure 2). But cannot bind to αVβ3-negative MCF7 cell line (Figure 3). We also use BBa_J36850 as a control. This strain cannot bind to αVβ3-positive MDA-MB-231 cell line (Figure 4) and αVβ3-negative MCF7 cell line (Figure 5). These results suggest that we successfully improve the part BBa_J36850. (Click here to see the method)
Figure 2. αVβ3-positive MDA-MB-231 cell line was incubated with E. coli which constructively expressed RFP and contained BBa_J36850. This improved part expressed RGD motif under the control of lac promoter.
Figure 3. αVβ3-negative MCF7 cell line was incubated with E. coli which constructive expressed RFP and contained BBa_J36850. This improved part expressed RGD motif under the control of lac promoter.
Figure 4. αVβ3-positive MDA-MB-231 cell line was incubated with E. coli which constructively expressed RFP and contained BBa_J36850.
Figure 5. αVβ3-negative MCF7 cell line was incubated with E. coli which constructively expressed RFP and contained BBa_J36850.
1. Grow Hela GSDMD KO cells in a humidified 37 °C, 5% CO2 tissue-culture incubator.
2. Count the cells using a hemocytometer. Seed in 24-well (5 × 10^4 per well) and grow overnight.
Preparation of Bacteria
1. Grow bacteria overnight 16 h in 2 mL LB in a 15-mL tube. Incubate at 37 °C in a shaking incubator (200 rpm).
2. Subculture bacteria by transferring 300 μL of the overnight culture into 5 mL of LB in a loosely capped 50-mL tube. Incubate at 37 °C in a shaking incubator (200 rpm) to late log phase.
3. Pellet 1 mL of the Salmonella subculture by centrifugation at 1000 g in a microfuge for 2 min at room temperature.
4. Remove 900 μL of supernatant and gently resuspend the pellet in 900 μL PBS.
Infection
1. Aspirate media and rinse the monolayer twice with PBS.
2. Inoculate cells with bacteria (MOI = 100) by adding bacteria directly to the cell-culture supernatant.
3. Incubate for 2 h at 37 °C in 5% CO2.
4. Aspirate media and wash
5. Add fresh GM containing 100 μg/mL gentamicin and 16 μg/mL incubate at 37 °C in 5% CO2 for 2 h.
6. Replace GM with fresh GM containing 20 μg/mL gentamicin for 1 h.
7.Add 16 μg/mL ATc for remainder of experiment.
Observation is taken after 5 min, 30 min, 1.5 h.
1 Park, S. H. et al. RGD Peptide Cell-Surface Display Enhances the Targeting and Therapeutic Efficacy of Attenuated Salmonella-mediated Cancer Therapy. Theranostics 6, 1672-1682, doi:10.7150/thno.16135 (2016).
