Difference between revisions of "Part:BBa K606014:Experience"
 
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= Characterization tRNA amber suppressor=
 
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==Protocol==
 
  
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In order to characterize the tRNA amber suppressor, as well as the pHyperspank, a special part was made: Pveg-SpoVG-GFP amber-TT.
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This part has been characterized under the control of the pT7 T7 RNA polymerase promoter (part K606035) in E. coli BL21 strain.
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A double transformation was done with minipreps of two plasmids of different antibiotic resistances : pSB1C3 holding the tRNA under the control of pHyperspank and pSB1AK3 holding the GFP with the amber codon under the control of a constitutive promotor. Two groups of cultures of each picked colony were made. One group was cultured in rich medium without IPTG whilst the other group was cultured in rich medium with IPTG (4mM in 10mL). A culture of the strain holding GFP amber alone was grown in parallel. The two tubes of each colony were compared along with the negative control (GFP amber alone). Cultures were then diluted for microscopy, glycerols, and future experiments. We used the following strains to characterize this system:
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'''GFP amber'''
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'''Fluorescence kinetics'''
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Strain holding GFPmut3B, mutated with a single amber mutation under the control of the constitutive promotor Pveg, in the plasmid pSB1AK3 (AmpR).
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'''tRNA amber suppressor'''
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<p>The measurements have been carried out on a spectrophotometer, at 37°C under transient shaking. The experiment lasted 4h, we tested several colonies and several IPTG concentrations. The OD 600nm and the fluorescence of the GFP (excitation: 470nm / emission:515 nm) was measured every 5 min, and the ratio of the two was calculated.</p>
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Strain holding the tRNA amber suppressor, under the control of IPTG inducible strong promotor pHyperspank, in the plasmid pSB1C3 (CmR).
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'''Double transformant'''
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<p>All values were normalized by substracting the fluorescence/OD value of the well with 0 mM IPTG at time 0. The values given are in arbitrary units.</p>
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Double transformant of the two previous constructs in their plasmids respectively.
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Two groups of cultures are grown: with or without IPTG.
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Direct observation under fluorescence lamp and microscopy was used to characterize this system.
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[[Image:GrowthpT7GFPt7ter.png|center|thumb|500px|Fig1: Growth curves for BL21 strain carrying the part]]
 
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<br>We have also <b><a href="http://2011.igem.org/Team:Paris_Bettencourt/Experiments/pHyperSpank">characterised the pHyperSpank promoter</a></b>, compatible with <i>B.subtilis</i> and which is used in this system.
 
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==Characterization of the tRNA biobrick (fluorescence lamp)==
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<p>After 2 hrs of induction, we see a clear increase of the fluorescence proportional to the IPTG concentration (that is to say with the quantity of T7 polymerase induced in the cell). After 4 hrs, the expression of GFP under the pT7 is still not saturated</p>
  
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The cultures of strains having transformed both tRNA amber suppressor and GFP amber were strongly fluorescent, while the culture of the GFP amber alone as well as the culture of the colony without IPTG induction showed basal fluorescence.
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[[Image:GFPamberwithtRNA.jpg|350px|thumb|center|On the left, the culture of the strain holding GFP amber alone under the control of Pveg (constitutive promotor). On the right, the culture of the double transformant with IPTG induction (4mM)]]
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[[Image:pT7GFPSaturated.png|center|thumb|600px|Fig2: Comparison of the Fluo/OD ratio for transcription]]
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==Characterization of the tRNA biobrick (microscopy)==
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<p>Here, we plot the ratio of induction of the T7 polymerase dependant construct for the different concentrations of IPTG at a given time (4 hrs), taking the well with 0 IPTG at time 0 as the reference.</p>
  
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{| border="1" class="wikitable" style="text-align: center;"
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|+GFP amber / E-coli at 37°C
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|[[Image:Trnaminus gfp amber trans 1.jpg|350px|thumb|center|E-Coli  at 37°C  (trans image)]]
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|[[Image:Trnaminus_gfp_amber_fluo_1.jpg|350px|thumb|center|E-Coli  at 37°C  (gfp image)]]
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|[[Image:Trnaminus gfp amber trans 2.jpg|350px|thumb|center|E-Coli  at 37°C  (trans image)]]
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|[[Image:Trnaminus gfp amber fluo 2.jpg|350px|thumb|center|E-Coli  at 37°C  (gfp image)]]
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|}
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{| border="1" class="wikitable" style="text-align: center;"
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|+GFP amber +tRNA amber -IPTG/ E-coli at 37°C
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|[[Image:Trna_noiptg_gfp_amber_trans_.jpg|350px|thumb|center|E-Coli  at 37°C  (trans image)]]
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|[[Image:Trna noiptg gfp amber fluo 1.jpg|350px|thumb|center|E-Coli  at 37°C  (gfp image)]]
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|[[Image:Trna noiptg gfp amber trans 2.jpg|350px|thumb|center|E-Coli  at 37°C  (trans image)]]
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|[[Image:Trna noiptg gfp amber fluo 2.jpg|350px|thumb|center|E-Coli  at 37°C  (gfp image)]]
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|}
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{| border="1" class="wikitable" style="text-align: center;"
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|+GFP amber +tRNA amber +IPTG/ E-coli at 37°C
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|[[Image:Trna iptg gfp amber trans 3.jpg|350px|thumb|center|E-Coli  induced with IPTG at 37°C  (trans image)]]
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|[[Image:Trna iptg gfp amber fluo 3.jpg|350px|thumb|center|E-Coli induced with IPTG at 37°C  (gfp image)]]
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|[[Image:Trna iptg gfp amber trans 4.jpg|350px|thumb|center|E-Coli induced with IPTG at 37°C  (trans image)]]
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|[[Image:Trna iptg gfp amber fluo 4.jpg|350px|thumb|center|E-Coli  induced with IPTG at 37°C  (gfp image)]]
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===Applications of BBa_K606014===
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===User Reviews===
 
===User Reviews===
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Latest revision as of 07:16, 27 October 2011


This part has been characterized under the control of the pT7 T7 RNA polymerase promoter (part K606035) in E. coli BL21 strain.

Fluorescence kinetics

The measurements have been carried out on a spectrophotometer, at 37°C under transient shaking. The experiment lasted 4h, we tested several colonies and several IPTG concentrations. The OD 600nm and the fluorescence of the GFP (excitation: 470nm / emission:515 nm) was measured every 5 min, and the ratio of the two was calculated.

All values were normalized by substracting the fluorescence/OD value of the well with 0 mM IPTG at time 0. The values given are in arbitrary units.

Fig1: Growth curves for BL21 strain carrying the part

After 2 hrs of induction, we see a clear increase of the fluorescence proportional to the IPTG concentration (that is to say with the quantity of T7 polymerase induced in the cell). After 4 hrs, the expression of GFP under the pT7 is still not saturated

Fig2: Comparison of the Fluo/OD ratio for transcription

Here, we plot the ratio of induction of the T7 polymerase dependant construct for the different concentrations of IPTG at a given time (4 hrs), taking the well with 0 IPTG at time 0 as the reference.

User Reviews

UNIQ9de83745ca029321-partinfo-00000002-QINU UNIQ9de83745ca029321-partinfo-00000003-QINU