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

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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K1632020 short</partinfo>
 
<partinfo>BBa_K1632020 short</partinfo>
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sequence confirmed
 
sequence confirmed
  
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===Source===
+
===Materials and Methods===
  
−
1.Construction
+
<b>1.Construction</b><br>
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All the samples were JM2.300 strain with antibiotic resistance to ampicillin and kanamycin.
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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)
+
(1) J23100_''rhlR''_TT_Plux_''CmR'' (pSB6A1) + Plac_''lasI'' (pSB3K3)<br>
−
B.Pcon_rhlR_TT_Plux_CmR (pSB6A1) +⊿P_lasI (pSB3K3)
+
(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
+
(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
+
(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)
+
(5) J23100_''rhlR''_TT_Plux_''CmRssrA'' (pSB6A1) + Plac_''lasI'' (pSB3K3)<br>
−
F.Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1) +⊿P_lasI (pSB3K3)
+
(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>
 +
 
 +
 
 +
<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>
 +
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.<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>
 +
<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 (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) + 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) + 100 mg/mL Chloramphenicol (3 microL)<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 0.9, dilute the cell medium to 1/5.)<br>
 +
 
 +
====Results====
 +
 
 +
[[Image:Tokyo_Tech Pcon_rbs_lsaR_TT_Plux_rbs_cmRssrA.png|thumb|center|550px|<b>Fig. 2.</b> The cells growth with <partinfo>BBa_K1632022</partinfo> in LB containing Cm]]
 +
[[Image:Tokyo_Tech Pcon_rbs_rhlR_TT_Plux_rbs_cmRssrA.png|thumb|center|550px|<b>Fig. 3.</b> The cells growth with <partinfo>BBa_K1632023</partinfo> in LB containing 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===
  
−
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.
+
Composite of BBa_K395160, BBa_M0052
−
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===
 +
1.Bo Hu et al. (2010) An Environment-Sensitive Synthetic Microbial Ecosystem. PLoS ONE 5(5): e10619

Latest revision as of 00:43, 19 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.)

Results

Fig. 2. The cells growth with BBa_K1632022 in LB containing Cm
Fig. 3. The cells growth with BBa_K1632023 in LB containing 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_K395160, BBa_M0052

References

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