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

(→‎Materials and Methods)
(→‎Materials and Methods)
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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>
  
−
A.Pcon_rhlR_TT_Plux_CmR (pSB6A1) + Plac_lasI (pSB3K3)<br>
+
(1) J23100_rhlR_TT_Plux_CmR (pSB6A1) + Plac_lasI (pSB3K3)<br>
−
B.Pcon_rhlR_TT_Plux_CmR (pSB6A1) + promoter less_lasI (pSB3K3)<br>
+
(2) J23100_rhlR_TT_Plux_CmR (pSB6A1) + promoter less_lasI (pSB3K3)<br>
−
C.Pcon_rhlR_TT_promoter less_CmR (pSB6A1) + Plac_lasI (pSB3K3)…Negative control #1<br>
+
(3) J23100_rhlR_TT_promoter less_CmR (pSB6A1) + Plac_lasI (pSB3K3)…Negative control #1<br>
−
D.Pcon_rhlR_TT_promoter less_CmR (pSB6A1) + promoter less_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) + promoter less_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>
Line 30: Line 30:
 
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>
−
<span style="margin-left: 20px;">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>
−
<span style="margin-left: 20px;">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>
−
<span style="margin-left: 20px;">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>
−
<span style="margin-left: 20px;">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 (30 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>
  
 
===Source===
 
===Source===

Revision as of 19:01, 17 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

Materials and Methods

1.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


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) + 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 (30 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.)

Source

Composite of BBa_K395160, BBa_M0052

References

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