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>
  
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A.Pcon_lasR_TT_Plux_CmR (pSB6A1) + Plac_rhlI (pSB3K3)<br>
+
(1) J23100_''lasR''_TT_Plux_''CmR'' (pSB6A1) + Plac_''rhlI'' (pSB3K3)<br>
−
B.Pcon_lasR_TT_Plux_CmR (pSB6A1) + promoter less_rhlI (pSB3K3)<br>
+
(2) J23100_''lasR''_TT_Plux_''CmR'' (pSB6A1) + promoter less_''rhlI'' (pSB3K3)<br>
−
C.Pcon_lasR_TT_⊿P_CmR (pSB6A1) + Plac_rhlI (pSB3K3)…Negative control #1<br>
+
(3) J23100_''lasR''_TT_promoter less_''CmR'' (pSB6A1) + Plac_''rhlI'' (pSB3K3)…Negative control #1<br>
−
D.Pcon_lasR_TT_⊿P_CmR (pSB6A1) + promoter less_rhlI (pSB3K3)…Negative control #2<br>
+
(4) J23100_''lasR''_TT_promoter less_''CmR'' (pSB6A1) + promoter less_''rhlI'' (pSB3K3)…Negative control #2<br>
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E.Pcon_lasR_TT_Plux_CmRssrA (pSB6A1) + Plac_rhlI (pSB3K3)<br>
+
(5) J23100_''lasR''_TT_Plux_''CmRssrA'' (pSB6A1) + Plac_''rhlI'' (pSB3K3)<br>
−
F.Pcon_lasR_TT_Plux_CmRssrA (pSB6A1) + promoter less_rhlI (pSB3K3)<br>
+
(6) J23100_''lasR''_TT_Plux_''CmRssrA'' (pSB6A1) + promoter less_''rhlI'' (pSB3K3)<br>
  
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[[Image:|thumb|center|600px|<b>Fig. 1. </b>Plasmids]]<br>
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<b>2.Assay protocol</b><br>
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+
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<b>2. Assay protocol</b><br>
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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>
 
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 Amp (50 microg/mL) and Kan (30 microg/mL) and grow the cells at 37°C until the observed OD590 reaches 0.5.<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>
 
3.Centrifuge 1 mL of the sample at 5000g, RT for 1 minute.<br>
−
4.Suspend the pellet in 1 mL 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) + DMSO (3 microL) + Chloramphenicol (6 microL of 25 microg/mL) + 99.5% ethanol (6 microL)<br>
+
<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>
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　　b)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (3 microL) + Chloramphenicol (9 microL of 25 microg/mL) + 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>
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　　c)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (3 microL) + Chloramphenicol (12 microL of 25 microg/mL)<br>
+
<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>
 +
<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 the optical density every hour. (If the optical density is over 0.9, dilute the cell medium to 1/5.)<br>
+
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 39: 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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 383
    Illegal AgeI site found at 580
  • 1000
    INCOMPATIBLE 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

Fig. 1. 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

Composite of BBa_K553003, BBa_K1632021

References

1.Bo Hu et al. (2010) An Environment-Sensitive Synthetic Microbial Ecosystem. PLoS ONE 5(5): e10619