Part:BBa_K1632022:Experience

Materials and Methods

1.Construction

All the samples were JM2.300 strain with antibiotic resistance to ampicillin and kanamycin.

(1) J23100_lasR_TT_Plux_CmR (pSB6A1) + Plac_rhlI (pSB3K3)
(2) J23100_lasR_TT_Plux_CmR (pSB6A1) + promoter less_rhlI (pSB3K3)
(3) J23100_lasR_TT_promoter less_CmR (pSB6A1) + Plac_rhlI (pSB3K3)…Negative control #1
(4) J23100_lasR_TT_promoter less_CmR (pSB6A1) + promoter less_rhlI (pSB3K3)…Negative control #2
(5) J23100_lasR_TT_Plux_CmRssrA (pSB6A1) + Plac_rhlI (pSB3K3)
(6) J23100_lasR_TT_Plux_CmRssrA (pSB6A1) + promoter less_rhlI (pSB3K3)

2.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) + 5 microL 3OC12HSL (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) + 5 microL 3OC12HSL (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.)

3.Results

Fig. 1. The cells growth with Cm

4.Discussion

The cells which have rbs-cmR without an ssrA degradation tag showed active growth. It showed leaky expression of CmR. Because of them, cells grew actively even in the absence of AHL. But compared with circuits without an ssrA tag, our improved BBa_K1632022 indeed showed much slower growth.
From the results above, we can say that the leaked CmR protein was degraded immediately because of the ssrA tag added right after the CmR protein. These results show the improved function of AHL-dependent CmR expression.

More information

For more information, see http://2015.igem.org/Team:Tokyo_Tech/Project Our work in Tokyo_Tech 2015 wiki, http://2015.igem.org/Team:Tokyo_Tech/Experiment/ssrA_tag_degradation_assay About ssrA-tag, http://2015.igem.org/Team:Tokyo_Tech/Experiment/Overview_of_fim_inversion_system About ''fim'' inversion system

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