Difference between revisions of "Part:BBa K1632021:Design"
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__NOTOC__ | __NOTOC__ | ||
<partinfo>BBa_K1632021 short</partinfo> | <partinfo>BBa_K1632021 short</partinfo> | ||
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===Materials and Methods=== | ===Materials and Methods=== | ||
+ | <b>1.Construction</b><br> | ||
+ | All the samples were JM2.300 strain with antibiotic resistance to ampicillin and kanamycin.<br> | ||
+ | |||
+ | (1) J23100_''rhlR''_TT_Plux_''CmR'' (pSB6A1) + Plac_''lasI'' (pSB3K3)<br> | ||
+ | (2) J23100_''rhlR''_TT_Plux_''CmR'' (pSB6A1) + promoter less_''lasI'' (pSB3K3)<br> | ||
+ | (3) J23100_''rhlR''_TT_promoter less_''CmR'' (pSB6A1) + Plac_''lasI'' (pSB3K3)…Negative control #1<br> | ||
+ | (4) J23100_''rhlR''_TT_promoter less_''CmR'' (pSB6A1) + promoter less_''lasI'' (pSB3K3)…Negative control #2<br> | ||
+ | (5) J23100_''rhlR''_TT_Plux_''CmRssrA'' (pSB6A1) + Plac_''lasI'' (pSB3K3)<br> | ||
+ | (6) J23100_''rhlR''_TT_Plux_''CmRssrA'' (pSB6A1) + promoter less_''lasI'' (pSB3K3)<br> | ||
+ | |||
+ | [[Image:RhlR cmRssrA Assay Construction.png|thumb|center|600px|<b>Fig. 1. </b>Plasmids]]<br> | ||
+ | |||
+ | |||
+ | <b>2.Assay protocol</b><br> | ||
+ | 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.<br> | ||
+ | 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.<br> | ||
+ | 3.Centrifuge 1 mL of the sample at 5000g, RT for 1 minute.<br> | ||
+ | 4.Suspend the pellet in 1mL of LB containing Amp and Kan.<br> | ||
+ | 5.Add 30 microL of suspension in the following medium.<br> | ||
+ | <span style="margin-left: 20px;">a)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + 500 microL C4HSL (3 microL) + 99.5% ethanol (3 microL)<br> | ||
+ | <span style="margin-left: 20px;">b)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (3 microL) + 99.5% ethanol (3 microL)<br> | ||
+ | <span style="margin-left: 20px;">c)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + 500 microL C4HSL (3 microL) + 100 mg/mL Chloramphenicol (3 microL)<br> | ||
+ | <span style="margin-left: 20px;">d)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (3 microL) + 100 mg/mL Chloramphenicol (3 microL)<br> | ||
+ | 6.Grow the samples of cells at 37°C for more than 8 hours.<br> | ||
+ | 7.Measure optical density every hour. (If the optical density is over 0.9, dilute the cell medium to 1/5.)<br> | ||
+ | |||
+ | ====Results==== | ||
+ | |||
+ | [[Image:Tokyo_Tech Pcon_rbs_lsaR_TT_Plux_rbs_cmRssrA.png|thumb|center|550px|<b>Fig. 2.</b> The cells growth with <partinfo>BBa_K1632022</partinfo> in LB containing Cm]] | ||
+ | [[Image:Tokyo_Tech Pcon_rbs_rhlR_TT_Plux_rbs_cmRssrA.png|thumb|center|550px|<b>Fig. 3.</b> The cells growth with <partinfo>BBa_K1632023</partinfo> in LB containing Cm]]<br> | ||
+ | |||
+ | ===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]] | ||
===Source=== | ===Source=== | ||
− | + | Composite of BBa_R0062, BBa_K1632020 | |
===References=== | ===References=== | ||
+ | 1.Bo Hu et al. (2010) An Environment-Sensitive Synthetic Microbial Ecosystem. PLoS ONE 5(5): e10619 |
Latest revision as of 00:45, 19 September 2015
Plux_rbs_CmRssrA
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
sequence confirmed
Materials and Methods
1.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) + promoter less_lasI (pSB3K3)
(3) J23100_rhlR_TT_promoter less_CmR (pSB6A1) + Plac_lasI (pSB3K3)…Negative control #1
(4) J23100_rhlR_TT_promoter less_CmR (pSB6A1) + promoter less_lasI (pSB3K3)…Negative control #2
(5) J23100_rhlR_TT_Plux_CmRssrA (pSB6A1) + Plac_lasI (pSB3K3)
(6) J23100_rhlR_TT_Plux_CmRssrA (pSB6A1) + promoter less_lasI (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) + 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.)
Results
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
Source
Composite of BBa_R0062, BBa_K1632020
References
1.Bo Hu et al. (2010) An Environment-Sensitive Synthetic Microbial Ecosystem. PLoS ONE 5(5): e10619