Difference between revisions of "Part:BBa K1632022:Design"
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__NOTOC__ | __NOTOC__ | ||
<partinfo>BBa_K1632022 short</partinfo> | <partinfo>BBa_K1632022 short</partinfo> | ||
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All the samples were JM2.300 strain with antibiotic resistance to ampicillin and kanamycin.<br> | All the samples were JM2.300 strain with antibiotic resistance to ampicillin and kanamycin.<br> | ||
− | + | (1) J23100_''lasR''_TT_Plux_''CmR'' (pSB6A1) + Plac_''rhlI'' (pSB3K3)<br> | |
− | + | (2) J23100_''lasR''_TT_Plux_''CmR'' (pSB6A1) + promoter less_''rhlI'' (pSB3K3)<br> | |
− | + | (3) J23100_''lasR''_TT_promoter less_''CmR'' (pSB6A1) + Plac_''rhlI'' (pSB3K3)…Negative control #1<br> | |
− | + | (4) J23100_''lasR''_TT_promoter less_''CmR'' (pSB6A1) + promoter less_''rhlI'' (pSB3K3)…Negative control #2<br> | |
− | + | (5) J23100_''lasR''_TT_Plux_''CmRssrA'' (pSB6A1) + Plac_''rhlI'' (pSB3K3)<br> | |
− | + | (6) J23100_''lasR''_TT_Plux_''CmRssrA'' (pSB6A1) + promoter less_''rhlI'' (pSB3K3)<br> | |
− | + | ||
− | + | ||
<b>2.Assay protocol</b><br> | <b>2.Assay protocol</b><br> | ||
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4.Suspend the pellet in 1mL of LB containing Amp and Kan.<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> | 5.Add 30 microL of suspension in the following medium.<br> | ||
− | a)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + | + | <span style="margin-left: 20px;">a)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + 5 microL 3OC12HSL (3 microL) + 99.5% ethanol (3 microL)<br> |
− | b)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO ( | + | <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> |
− | c)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + | + | <span style="margin-left: 20px;">c)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + 5 microL 3OC12HSL (3 microL) + 100 mg/mL Chloramphenicol (3 microL)<br> |
− | d)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO ( | + | <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> | 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 | + | 7.Measure optical density every hour. (If the optical density is over 0.9, dilute the cell medium to 1/5.)<br> |
+ | |||
+ | <b>3.Results</b> | ||
+ | |||
+ | [[Image:Tokyo_Tech Pcon_rbs_lsaR_TT_Plux_rbs_cmRssrA.png|thumb|center|550px|<b>Fig. 1.</b> The cells growth with Cm]] | ||
+ | |||
+ | ===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=== | ||
Line 40: | Line 45: | ||
===References=== | ===References=== | ||
+ | |||
+ | 1.Bo Hu et al. (2010) An Environment-Sensitive Synthetic Microbial Ecosystem. PLoS ONE 5(5): e10619 |
Latest revision as of 02:46, 19 September 2015
J23100_rbs_lasR_TT_Plux_rbs_CmRssrA
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 383
Illegal AgeI site found at 580 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 927
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_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
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_K553003, BBa_K1632021
References
1.Bo Hu et al. (2010) An Environment-Sensitive Synthetic Microbial Ecosystem. PLoS ONE 5(5): e10619