Difference between revisions of "Part:BBa R0011:Experience"

Line 79: Line 79:
 
UT-Tokyo 2011 Team has characterized this part through the test of our [https://parts.igem.org/Part:BBa_K518002 Firefly-Renilla Dual Luciferase Assay Kit]. We succesfully evaluated the relative expression levels of [https://parts.igem.org/Part:BBa_R0011 BBa_R0011] with various IPTG concentration, compared to that of [https://parts.igem.org/Part:BBa_J23119 BBa_J23119] as a control. For experimental details, see [http://2011.igem.org/Team:UT-Tokyo our page].
 
UT-Tokyo 2011 Team has characterized this part through the test of our [https://parts.igem.org/Part:BBa_K518002 Firefly-Renilla Dual Luciferase Assay Kit]. We succesfully evaluated the relative expression levels of [https://parts.igem.org/Part:BBa_R0011 BBa_R0011] with various IPTG concentration, compared to that of [https://parts.igem.org/Part:BBa_J23119 BBa_J23119] as a control. For experimental details, see [http://2011.igem.org/Team:UT-Tokyo our page].
 
|};
 
|};
 +
 +
{|width='80%' style='border:1px solid gray'
 +
|-
 +
|width='10%'|
 +
<partinfo>BBa_K592008 AddReview 5</partinfo>
 +
<I>[http://2012.igem.org/Team:Uppsala_University Uppsala University 2012]</I>
 +
|width='60%' valign='top'|
 +
 +
'''iGEM Team Uppsala University 2012'''
 +
 +
'''Promoter strength'''
 +
 +
[[Image:Promotertestuppsala2012.png|300px|thumb|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 (<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.
 +
 +
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.
 +
 +
'''LacI repression'''
 +
 +
The possibility of repression by lacI and induction by IPTG was evaluated in the pSB4C15Iq backbone. Cells grown with IPTG (0.5 mM) had a 100-fold increase of RFP expression, when compared to those grown without. Read about [https://parts.igem.org/Part:BBa_K864009:Experience pSB4C15Iq] for details.
 +
 +
|}
  
  
 
<!-- DON'T DELETE --><partinfo>BBa_R0011 EndReviews</partinfo>
 
<!-- DON'T DELETE --><partinfo>BBa_R0011 EndReviews</partinfo>

Revision as of 17:25, 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_R0011

User Reviews

UNIQ3420fc02c866d109-partinfo-00000000-QINU

•••••

Antiquity

This review comes from the old result system and indicates that this part worked in some test.

UNIPV-Pavia iGEM 2009

UNIPV-Pavia iGEM 2009's Experience: BBa_R0011 - Plac hybrid promoter

•••

UNIPV-Pavia iGEM 2010

UNIPV-Pavia iGEM 2010's Experience

Washington 2010

Washington 2010's Experience: R0011 in different plasmids

••••

iGEM Kyoto 2010

We measured RPUs of R0011 in low number copy plasmid with various concentrations of IPTG

See details.

••••

iGEM11_Uppsala-Sweden

We measured RPUs of different promoters in low copy number plasmids.

See details.

;
•••••

UT-Tokyo 2011

UT-Tokyo 2011 Team has characterized this part through the test of our Firefly-Renilla Dual Luciferase Assay Kit. We succesfully evaluated the relative expression levels of BBa_R0011 with various IPTG concentration, compared to that of BBa_J23119 as a control. For experimental details, see [http://2011.igem.org/Team:UT-Tokyo our page].

;
•••••

[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.

LacI repression

The possibility of repression by lacI and induction by IPTG was evaluated in the pSB4C15Iq backbone. Cells grown with IPTG (0.5 mM) had a 100-fold increase of RFP expression, when compared to those grown without. Read about pSB4C15Iq for details.


UNIQ3420fc02c866d109-partinfo-00000009-QINU