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

(→‎Source)
(→‎Source)
Line 14: Line 14:
 
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.
  
−
A.Pcon_rhlR_TT_Plux_CmR (pSB6A1) + Plac_lasI (pSB3K3)  
+
A.Pcon_rhlR_TT_Plux_CmR (pSB6A1) + Plac_lasI (pSB3K3)<br>
−
B.Pcon_rhlR_TT_Plux_CmR (pSB6A1) +⊿P_lasI (pSB3K3)  
+
B.Pcon_rhlR_TT_Plux_CmR (pSB6A1) +⊿P_lasI (pSB3K3)<br>
−
C.Pcon_rhlR_TT_⊿P_CmR (pSB6A1) + Plac_lasI (pSB3K3)…Negative control #1  
+
C.Pcon_rhlR_TT_⊿P_CmR (pSB6A1) + Plac_lasI (pSB3K3)…Negative control #1<br>
−
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<br>
−
E.Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1) + Plac_lasI (pSB3K3)  
+
E.Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1) + Plac_lasI (pSB3K3)<br>
−
F.Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1) +⊿P_lasI (pSB3K3)  
+
F.Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1) +⊿P_lasI (pSB3K3)<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.  
+
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.  
+
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.  
+
3.Centrifuge 1 mL of the sample at 5000g, RT for 1 minute.<br>
−
4.Suspend the pellet in 1mL of LB containing Amp and Kan.  
+
4.Suspend the pellet in 1mL of LB containing Amp and Kan.<br>
−
5.Add 30 microL of suspension in the following medium.  
+
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)
+
a)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + 50 microL C4HSL (30 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)
+
b)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (30 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)
+
c)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + 50 microL C4HSL (30 microL) + Chloramphenicol (100 microg/mL)<br>
−
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)<br>
−
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.<br>
−
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.)<br>
  
 
===References===
 
===References===

Revision as of 07:19, 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