Difference between revisions of "Part:BBa K1632020:Design"

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All the samples were JM2.300 strain with antibiotic resistance to ampicillin and kanamycin.
 
All the samples were JM2.300 strain with antibiotic resistance to ampicillin and kanamycin.
  
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A.Pcon_rhlR_TT_Plux_CmR (pSB6A1) + Plac_lasI (pSB3K3)
+
A.Pcon_rhlR_TT_Plux_CmR (pSB6A1) + Plac_lasI (pSB3K3)  
−
B.Pcon_rhlR_TT_Plux_CmR (pSB6A1) +⊿P_lasI (pSB3K3)
+
B.Pcon_rhlR_TT_Plux_CmR (pSB6A1) +⊿P_lasI (pSB3K3)  
−
C.Pcon_rhlR_TT_⊿P_CmR (pSB6A1) + Plac_lasI (pSB3K3)…Negative control #1
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C.Pcon_rhlR_TT_⊿P_CmR (pSB6A1) + Plac_lasI (pSB3K3)…Negative control #1  
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D.Pcon_rhlR_TT_⊿P_CmR (pSB6A1) +⊿P_lasI (pSB3K3)…Negative control #2
+
D.Pcon_rhlR_TT_⊿P_CmR (pSB6A1) +⊿P_lasI (pSB3K3)…Negative control #2  
−
E.Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1) + Plac_lasI (pSB3K3)
+
E.Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1) + Plac_lasI (pSB3K3)  
−
F.Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1) +⊿P_lasI (pSB3K3)
+
F.Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1) +⊿P_lasI (pSB3K3)  
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+
   
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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.
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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.  
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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.
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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.  
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3.Centrifuge 1 mL of the sample at 5000g, RT for 1 minute.
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3.Centrifuge 1 mL of the sample at 5000g, RT for 1 minute.  
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4.Suspend the pellet in 1mL of LB containing Amp and Kan.
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4.Suspend the pellet in 1mL of LB containing Amp and Kan.  
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5.Add 30 microL of suspension in the following medium.
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5.Add 30 microL of suspension in the following medium.  
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a)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + 50 microL C4HSL (30 microL) + 99.5% ethanol (3 microL)  
+
a)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + 50 microL C4HSL (30 microL) + 99.5% ethanol (3 microL)   
−
  b)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (30 microL) + 99.5% ethanol (3 microL)  
+
b)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (30 microL) + 99.5% ethanol (3 microL)   
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  c)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + 50 microL C4HSL (30 microL) + Chloramphenicol (100 microg/mL)
+
c)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + 50 microL C4HSL (30 microL) + Chloramphenicol (100 microg/mL)   
−
  d)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (30 microL) + Chloramphenicol (100 microg/mL)
+
d)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (30 microL) + Chloramphenicol (100 microg/mL)   
−
  6.Grow the samples of cells at 37°C for more than 8 hours.
+
6.Grow the samples of cells at 37°C for more than 8 hours.  
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7.Measure optical density every hour. (If the optical density is over 1.0, dilute the cell medium to 1/5.)
+
7.Measure optical density every hour. (If the optical density is over 1.0, dilute the cell medium to 1/5.)
  
 
===References===
 
===References===

Revision as of 07:16, 12 September 2015


rbs_CmR(ssrA degradation tag)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

sequence confirmed

Source

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

A.Pcon_rhlR_TT_Plux_CmR (pSB6A1) + Plac_lasI (pSB3K3) B.Pcon_rhlR_TT_Plux_CmR (pSB6A1) +⊿P_lasI (pSB3K3) C.Pcon_rhlR_TT_⊿P_CmR (pSB6A1) + Plac_lasI (pSB3K3)…Negative control #1 D.Pcon_rhlR_TT_⊿P_CmR (pSB6A1) +⊿P_lasI (pSB3K3)…Negative control #2 E.Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1) + Plac_lasI (pSB3K3) F.Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1) +⊿P_lasI (pSB3K3)

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) + 50 microL C4HSL (30 microL) + 99.5% ethanol (3 microL) b)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (30 microL) + 99.5% ethanol (3 microL) c)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + 50 microL C4HSL (30 microL) + Chloramphenicol (100 microg/mL) d)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (30 microL) + Chloramphenicol (100 microg/mL) 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 1.0, dilute the cell medium to 1/5.)

References