Difference between revisions of "Part:BBa K1632023:Experience"
 
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__TOC__
 
__TOC__
 
===Materials and Methods===
 
===Materials and Methods===
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<b>1.Construction</b><br>
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 +
====Construction====
 +
 
 
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>
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(1) J23100_''rhlR''_TT_Plux_''CmR'' (pSB6A1) + Plac_''lasI'' (pSB3K3)<br>
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B.Pcon_rhlR_TT_Plux_CmR (pSB6A1) +⊿P_lasI (pSB3K3)<br>
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(2) J23100_''rhlR''_TT_Plux_''CmR'' (pSB6A1) +promoter_''lasI'' (pSB3K3)<br>
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C.Pcon_rhlR_TT_⊿P_CmR (pSB6A1) + Plac_lasI (pSB3K3)…Negative control #1<br>
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(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<br>
+
(4) J23100_''rhlR''_TT_promoter less_''CmR'' (pSB6A1) +promoter_''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) +⊿P_lasI (pSB3K3)<br>
+
(6) J23100_''rhlR''_TT_Plux_''CmRssrA'' (pSB6A1) +promoter_''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>
  
 +
====Assay protocol====
  
−
<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>
 
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>
 
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>
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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>
−
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>
−
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>
−
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>
−
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 (3 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>
 +
 
 +
====Results====
 +
 
 +
[[Image:Tokyo_Tech Pcon_rbs_rhlR_TT_Plux_rbs_cmRssrA.png|thumb|center|550px|<b>Fig. 2.</b> The cells growth with Cm]]<br>
 +
 
 +
====Discussion====
 +
 
 +
<span style="margin-left: 10px;">The cells which have ''rbs-cmR'' without an ssrA degradation tag showed active growth. It showed leaky expression of CmR. Because of them, cells grew actively even in the absence of AHL. But compared with circuits without an ssrA tag, our improved BBa_K1632023 indeed showed much slower growth.<br>
 +
<span style="margin-left: 10px;">From the results above, we can say that the leaked CmR protein was degraded immediately because of the ssrA tag added right after the CmR protein.  These results show the improved function of AHL-dependent CmR expression.
 +
 
 +
===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]]
  
 
===Applications of BBa_K1632023===
 
===Applications of BBa_K1632023===

Latest revision as of 00:48, 19 September 2015

J23100_rbs_rhlR_TT_Plux_rbs_CmRssrA

Materials and Methods

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_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_lasI (pSB3K3)…Negative control #2
(5) J23100_rhlR_TT_Plux_CmRssrA (pSB6A1) + Plac_lasI (pSB3K3)
(6) J23100_rhlR_TT_Plux_CmRssrA (pSB6A1) +promoter_lasI (pSB3K3)

Fig. 1. Plasmids

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 (3 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 Cm

Discussion

The cells which have rbs-cmR without an ssrA degradation tag showed active growth. It showed leaky expression of CmR. Because of them, cells grew actively even in the absence of AHL. But compared with circuits without an ssrA tag, our improved BBa_K1632023 indeed showed much slower growth.
From the results above, we can say that the leaked CmR protein was degraded immediately because of the ssrA tag added right after the CmR protein. These results show the improved function of AHL-dependent CmR expression.

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

Applications of BBa_K1632023

User Reviews

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