Difference between revisions of "Part:BBa I14032:Experience"

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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 
This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
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This review comes from the old result system and indicates that this part did not work in some test.
 
This review comes from the old result system and indicates that this part did not work in some test.
 
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{|width='80%' style='border:1px solid gray'
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<partinfo>BBa_K592008 AddReview 5</partinfo>
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<I>[http://2012.igem.org/Team:Uppsala_University Uppsala University 2012]</I>
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|width='60%' valign='top'|
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'''iGEM Team Uppsala University 2012'''
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'''Promoter strength'''
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[[Image:Promotertestuppsala2012.png|300px|thumb|Promoter strenghts in MG1566 and DH5alpha, relative to J23101.]]
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A promoter test was carried out to put synthetic and natural promoters on the same scale. Every promoter was assembled before B0032-SYFP2 (<partinfo>K864101</partinfo>) in the backbone <partinfo>K592200</partinfo> (very similar to the pSB3x5 backbones). The test was performed in E coli expression strain MG1655 and cloning strain DH5alpha, by flow cytometry fluorescence measurements of single cells.
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Triplicates of each strain and promoter were inoculated in 2 mL LB media with spectinomycin (50 µg/mL) and grown overnight shaking at 37° C. Samples were equilibrated in PBS solution at 1:160 dilution for one hour, and then measured by a BD Biosciences FACSaria III. 10^5 cells of each sample were individually measured and averaged, with dead and other non-flourescent cells excluded. Promoter strength is noted as fractions of the reference promoter's, J23101, strength in corresponding strain.
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{|
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|
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!MG1566
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!DH5alpha
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|-
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|J23101
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|1
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|1
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|-
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|J23106
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|0.19
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|0.37
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|-
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|J23110
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|0.27
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|0.50
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|-
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|J23116
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|N/A
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|0.11
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|-
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|Plac
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|0.34
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|0.67
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|-
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|PlacIq
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|0.03
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|0.05
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|-
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|T5lac
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|0.217
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|0.54
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|-
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|PLlacO
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|0.87
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|N/A
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|}
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The variance in expression between MG1655 and DH5α may depend on the reference J23101. The maximum protein expression may be lower in DH5α, due to its lower fitness resulting in lower expression of SYFP2 in the J23101 construct. Alternatively, the clone with J23101 in DH5α may have been weaker than average, resulting in higher RPU values compared to other DH5α.
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|};
 
<!-- DON'T DELETE --><partinfo>BBa_I14032 EndReviews</partinfo>
 
<!-- DON'T DELETE --><partinfo>BBa_I14032 EndReviews</partinfo>

Latest revision as of 16:48, 18 October 2012

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_I14032

Despite this part being classified under "repressible" promoters - it is a constitutive promoter. I originally mistook this part for the lac promoter - which is instead R0010 -- wtimp0

User Reviews

UNIQc9a2eaf1968c544e-partinfo-00000000-QINU

Antiquity

This review comes from the old result system and indicates that this part did not work in some test.

•••••

[http://2012.igem.org/Team:Uppsala_University Uppsala University 2012]

iGEM Team Uppsala University 2012

Promoter strength

Promoter strenghts in MG1566 and DH5alpha, relative to J23101.

A promoter test was carried out to put synthetic and natural promoters on the same scale. Every promoter was assembled before B0032-SYFP2 (BBa_K864101) in the backbone BBa_K592200 (very similar to the pSB3x5 backbones). The test was performed in E coli expression strain MG1655 and cloning strain DH5alpha, by flow cytometry fluorescence measurements of single cells.

Triplicates of each strain and promoter were inoculated in 2 mL LB media with spectinomycin (50 µg/mL) and grown overnight shaking at 37° C. Samples were equilibrated in PBS solution at 1:160 dilution for one hour, and then measured by a BD Biosciences FACSaria III. 10^5 cells of each sample were individually measured and averaged, with dead and other non-flourescent cells excluded. Promoter strength is noted as fractions of the reference promoter's, J23101, strength in corresponding strain.

MG1566 DH5alpha
J23101 1 1
J23106 0.19 0.37
J23110 0.27 0.50
J23116 N/A 0.11
Plac 0.34 0.67
PlacIq 0.03 0.05
T5lac 0.217 0.54
PLlacO 0.87 N/A

The variance in expression between MG1655 and DH5α may depend on the reference J23101. The maximum protein expression may be lower in DH5α, due to its lower fitness resulting in lower expression of SYFP2 in the J23101 construct. Alternatively, the clone with J23101 in DH5α may have been weaker than average, resulting in higher RPU values compared to other DH5α.

;

UNIQc9a2eaf1968c544e-partinfo-00000005-QINU