Part:BBa_K1632023:Design
J23100_rbs_rhlR_TT_Plux_rbs_CmRssrA
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 301
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 776
Illegal BsaI.rc site found at 933
Design Notes
sequence confirmed
Materials and Methods
Construction
All the samples were JM2.300 strain with antibiotic resistance to ampicillin and kanamycin.
(1) J23100_rhlR_TT_Plux_CmR (pSB6A1) + Plac_lasI (pSB3K3)
(2) J23100_rhlR_TT_Plux_CmR (pSB6A1) + promotor less_lasI (pSB3K3)
(3) J23100_rhlR_TT_promotor less_CmR (pSB6A1) + Plac_lasI (pSB3K3)…Negative control #1
(4) J23100_rhlR_TT_promotor less_CmR (pSB6A1) + promotor less_lasI (pSB3K3)…Negative control #2
(5) J23100_rhlR_TT_Plux_CmRssrA (pSB6A1) + Plac_lasI (pSB3K3)
(6) J23100_rhlR_TT_Plux_CmRssrA (pSB6A1) + promotor less_lasI (pSB3K3)
Assay protocol
1.Prepare overnight cultures for the samples in 3 mL LB medium, containing ampicillin (50 microg/mL) and kanamycin (30 microg/mL) at 37°C for 12 hours.
2.Make a 1:100 dilution in 3 mL of fresh LB containing antibiotic and grow the cells at 37°C until the observed OD590 reaches 0.5.
3.Centrifuge 1 mL of the sample at 5000g, RT for 1 minute.
4.Suspend the pellet in 1mL of LB containing Amp and Kan.
5.Add 30 microL of suspension in the following medium.
a)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + 500 microL C4HSL (3 microL) + 99.5% ethanol (3 microL)
b)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (3 microL) + 99.5% ethanol (3 microL)
c)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + 500 microL C4HSL (3 microL) + 100 mg/mL Chloramphenicol (3 microL)
d)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (3 microL) + 100 mg/mL Chloramphenicol (3 microL)
6.Grow the samples of cells at 37°C for more than 8 hours.
7.Measure optical density every hour. (If the optical density is over 0.9, dilute the cell medium to 1/5.)
Source
Composite of BBa_J23100, BBa_I1466, BBa_K1632021
References
1.Bo Hu et al. (2010) An Environment-Sensitive Synthetic Microbial Ecosystem. PLoS ONE 5(5): e10619