Difference between revisions of "Part:BBa J23116:Experience"

 
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<partinfo>BBa_J23116 AddReview 5</partinfo>
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<I>Username</I>
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<I>UNIPV-Pavia iGEM 2010</I>
 
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The <partinfo>BBa_J23100</partinfo>, <partinfo>BBa_J23101</partinfo>, <partinfo>BBa_J23105</partinfo>, <partinfo>BBa_J23106</partinfo>, <partinfo>BBa_J23110</partinfo>, <partinfo>BBa_J23114</partinfo>, <partinfo>BBa_J23116</partinfo>, <partinfo>BBa_J23118</partinfo> were charcterized in LB and M9 supplemented with glycerol (0.4%) growth media in high copy and low copy vectors in ''E. coli'' TOP10 (<partinfo>BBa_V1009</partinfo>).
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RPU and doubling time were characterized for all of them, according to the protocols reported in [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for RPU evaluation|this section]].
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The following measurement systems were used for high copy plasmids:
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*<partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23100</partinfo>
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*<partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23101</partinfo>
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*<partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23105</partinfo>
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*<partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23106</partinfo>
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*<partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23110</partinfo>
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*<partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23114</partinfo>
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*<partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23116</partinfo>
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*<partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23118</partinfo>
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In order to build low copy plasmid measurement systems, the EcoRI-PstI fragment (J231xx-RFP) of each <partinfo>BBa_J61002</partinfo>-BBa_J231xx was assembled into <partinfo>pSB4C5</partinfo> vector. This fragment contains the constitutive promoter of interest upstream a RBS-RFP-TT expression system.
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The following measurement parts were used for low copy plasmids:
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*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J23100</partinfo>
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*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J23101</partinfo>
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*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J23105</partinfo>
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*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J23106</partinfo>
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*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J23110</partinfo>
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*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J23114</partinfo>
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*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J23116</partinfo>
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*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J23118</partinfo>
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The RPU values and doubling times are here reported:
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{| align='center'
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|[[Image:pv_RPU_HC_LB.png|330px|thumb|center|Figure 5 - R.P.U. of the studied promoters from Anderson promoters' collection, LB medium and high copy plasmid (<partinfo>BBa_J61002</partinfo>) ]]||[[Image:pv_RPU_HC_M9.png|330px|thumb|center|Figure 6 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and high copy plasmid (<partinfo>BBa_J61002</partinfo>)]]
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|}
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{| align='center'
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|[[Image:pv_RPU_LC_M9.png|330px|thumb|center|Figure 7 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and low copy plasmid (<partinfo>pSB4C5</partinfo>). These plasmids were constructed by assembling the EcoRI-PstI fragment of <partinfo>BBa_J61002</partinfo>-BBa_J231xx in <partinfo>pSB4C5</partinfo> vector, in order to transfer the promoter and the RBS-RFP-TT expression construct from <partinfo>BBa_J61002</partinfo> to <partinfo>pSB4C5</partinfo>.]]
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|}
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The error bars represent the standard deviation for three dfferent wells in the same experiment.
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Doubling times were evaluated for the described cultures (HC stands for High Copy and LC stands for Low Copy):
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{| align='center' border='1'
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|rowspan='2'|<b>Promoter</b>
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|colspan='3'| <b>doubling time [minutes]</b>
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|-
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| LB in HC plasmid || M9 in HC plasmid || M9 in LC plasmid
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|-
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|<partinfo>BBa_J23100</partinfo> || 33.75 <br> ± <br> 1.34 || 82.53 <br> ± <br> 2.45 || 86.11 <br> ± <br> 4.45
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|-
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|<partinfo>BBa_J23101</partinfo> || 35.93 <br> ± <br> 0.62 || 82.68 <br> ± <br> 1.84 || 86.42 <br> ± <br> 1.91
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|-
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|<partinfo>BBa_J23105</partinfo> || 29.86 <br> ± <br> 0.33 || 63.09 <br> ± <br> 7.08 || 85.00 <br> ± <br> 5.13
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|-
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|<partinfo>BBa_J23106</partinfo> || 29.17 <br> ± <br> 0.96 || 68.11 <br> ± <br> 4.25 || 88.71 <br> ± <br> 0.90
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|-
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|<partinfo>BBa_J23110</partinfo> || 31.28 <br> ± <br> 0.42 || 67.52 <br> ± <br> 5.87 || 76.15 <br> ± <br> 2.16
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|-
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|<partinfo>BBa_J23114</partinfo> || 28.97 <br> ± <br> 0.49 || 59.44 <br> ± <br> 5.20 || 80.12 <br> ± <br> 0.95
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|-
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|<partinfo>BBa_J23116</partinfo> || 28.14 <br> ± <br> 0.25 || 72.74 <br> ± <br> 0.37 || 81.68 <br> ± <br> 3.08
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|-
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|<partinfo>BBa_J23118</partinfo> || 32.84 <br> ± <br> 0.31 || 73.64 <br> ± <br> 2.41 || 89.86 <br> ± <br> 2.93
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|}
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It was not possible to evaluate promoters activities in low copy number plasmids in LB because the RFP activity was too weak and not distinguishable from the background.
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'''Discussion''': we observed that the ranking previously documented in the Registry is not valid in all the tested conditions, even if a general agreement can be observed. As an example, <partinfo>BBa_J23110</partinfo> in high copy plasmid is stronger than <partinfo>BBa_J23118</partinfo>, in contrast with the ranking reported in the Registry.
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===Microplate reader experiments for constitutive promoters (R.P.U. evaluation)===
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*8 ul of long term storage glycerol stock were inoculated in 5 ml of LB or M9 + suitable antibiotic in a 15 ml falcon tube and incubated at 37°C, 220 rpm for about 16 hours.
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*The grown cultures were then diluted 1:100 in 5 ml of LB or M9 supplemented medium and incubated in the same conditions as before for about 4 hours.
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*These new cultures were diluted to an O.D.600 of 0.02 (measured with a TECAN F200 microplate reader on a 200 ul of volume per well; it is not equivalent to the 1 cm pathlength cuvette) in 2 ml (wanted final volume) LB or M9 + suitable antibiotic. In order to have the cultures at the desired O.D.600 (O.D._wanted=0.02), the following dilution was performed:
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[[Image:UNIPV_Pavia_OD600_dil.png|500px|center]]
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*These new dilutions were aliquoted in a flat-bottom 96-well microplate, avoiding to perform dynamic experiments in the microplate frame (in order to prevent evaporation effects in the frame). All the wells were filled with a 200 ul volume.
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*The microplate was incubated in the Tecan Infinite F200 microplate reader and fluorescence and absorbance were measured with this automatic protocol:
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**37°C constant for all the experiment;
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**sampling time of 5 minutes;
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**fluorescence gain of 50 or 70;
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**O.D. filter was 600 nm;
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**GFP filters were 485nm (ex) / 540nm (em);
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**RFP filters were 535nm (ex) / 620nm (em);
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**15 seconds of linear shaking (3mm amplitude) followed by 10 seconds of waiting before the measurements in order to make a homogeneous culture.
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**Experiment duration time: about 6 hours.
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===Data analysis for RPU evaluation===
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The RPUs are standard units proposed by  Kelly J. et al., 2009, in which the relative transcriptional strength of a promoter can be measured using a reference standard.
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RPUs have been computed as:
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[[Image:UNIPV_Pavia_RPU_formula.png|400px|center]]
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in which:
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* phi is the promoter of interest and J23101 is the reference standard promoter (taken from the Anderson Promoter Collection);
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* F is the blanked fluorescence of the culture, computed by subtracting for each time sample the fluorescence value of a negative control (a non-fluorescent culture). In our experiments, the TOP10 cells bearing BBa_B0032 or BBa_B0033 were usually used because they are RBSs and do not have expression systems for reporter genes;
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* ASB is the blanked absorbance (O.D.600) of the culture, computed as described in "Preliminary remarks" section.
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RPU measurement has the following advantages (under suitable conditions)
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* it is proportional to PoPS (Polymerase Per Second), a very important parameter that expresses the transcription rate of a promoter;
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* it uses a reference standard and so measurements can be compared between different laboratories.
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The hypotheses on which RPU theory is based can be found in Kelly J. et al., 2008, as well as all the mathematical steps. From our point of view, the main hypotheses that have to be satisfied are the following:
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* the reporter protein must have a half life higher than the experiment duration (we use GFPmut3 - <partinfo>BBa_E0040</partinfo> -, which has an estimated half life of at least 24 hours, or an engineered RFP - <partinfo>BBa_E1010</partinfo>, for which the half life has not been measured, but is qualitatively comparable with the GFP's);
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* strain, plasmid copy number, antibiotic, growth medium, growth conditions, protein generator assembled downstream of the promoter must be the same in the promoter of interest and in J23101 reference standard.
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* steady state must be valid, so (dF/dt)/ASB (proportional to the GFP synthesis rate per cell) must be constant.
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In order to compute the RPUs, the Scell signals ((dGFP/dt)/ASB)) of the promoter of interest and of the reference J23101 were averaged in the time interval corresponding to the exponential growth phase. The boundaries of exponential phase were identified with a visual inspection of the linear phase of the logarithmic growth curve.
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Revision as of 22:07, 27 October 2010

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