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

(→‎Design Notes)
(→‎Source)
Line 11: Line 11:
 
===Source===
 
===Source===
  
−
a
+
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===
 
===References===

Revision as of 07:11, 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