Difference between revisions of "Part:BBa K1632023:Design"

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===Materials and Methods===
 
===Materials and Methods===
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<b>1.Construction</b><br>
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 +
====Construction====
 +
 
 
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>
  
−
A.Pcon_rhlR_TT_Plux_CmR (pSB6A1) + Plac_lasI (pSB3K3)<br>
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(1) J23100_''rhlR''_TT_Plux_''CmR'' (pSB6A1) + Plac_''lasI'' (pSB3K3)<br>
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B.Pcon_rhlR_TT_Plux_CmR (pSB6A1) +⊿P_lasI (pSB3K3)<br>
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(2) J23100_''rhlR''_TT_Plux_''CmR'' (pSB6A1) + promoter less_''lasI'' (pSB3K3)<br>
−
C.Pcon_rhlR_TT_⊿P_CmR (pSB6A1) + Plac_lasI (pSB3K3)…Negative control #1<br>
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(3) J23100_''rhlR''_TT_promoter less_''CmR'' (pSB6A1) + Plac_''lasI'' (pSB3K3)…Negative control #1<br>
−
D.Pcon_rhlR_TT_⊿P_CmR (pSB6A1) +⊿P_lasI (pSB3K3)…Negative control #2<br>
+
(4) J23100_''rhlR''_TT_promoter less_''CmR'' (pSB6A1) + promoter less_''lasI'' (pSB3K3)…Negative control #2<br>
−
E.Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1) + Plac_lasI (pSB3K3)<br>
+
(5) J23100_''rhlR''_TT_Plux_''CmRssrA'' (pSB6A1) + Plac_''lasI'' (pSB3K3)<br>
−
F.Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1) +⊿P_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>
 
[[Image:RhlR cmRssrA Assay Construction.png|thumb|center|600px|<b>Fig. 1. </b>Plasmids]]<br>
  
 +
====Assay protocol====
  
−
<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>
 
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>
 
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>
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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) + 50 microL C4HSL (30 microL) + 99.5% ethanol (3 microL)<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>
−
b)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (30 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>
−
c)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + 50 microL C4HSL (30 microL) + Chloramphenicol (100 microg/mL)<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>
−
d)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (30 microL) + Chloramphenicol (100 microg/mL)<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 optical density every hour. (If the optical density is over 1.0, 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>
 +
 
 +
====Results====
 +
 
 +
[[Image:Tokyo_Tech Pcon_rbs_rhlR_TT_Plux_rbs_cmRssrA.png|thumb|center|550px|<b>Fig. 2.</b> The cells growth with 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===

Latest revision as of 00:47, 19 September 2015

J23100_rbs_rhlR_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
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 301
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE 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) + 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)

Fig. 1. Plasmids

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

Fig. 2. 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_J23100, BBa_I1466, BBa_K1632021

References

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