Difference between revisions of "Part:BBa K823036"

 
(13 intermediate revisions by 3 users not shown)
Line 2: Line 2:
 
<partinfo>BBa_K823036 short</partinfo>
 
<partinfo>BBa_K823036 short</partinfo>
  
cMyc-tag with RBS in Freiburg standard.
+
cMyc-tag with RBS in [[Help:Assembly_standard_25|Freiburg standard]].
 
+
  
 +
Find out more about the design of our [https://static.igem.org/mediawiki/parts/b/b9/LMU-Munich_2012_Our_Freiburg_standard_FusionPrefix.pdf prefix with ribosome binding site].
  
 
prefix:GAATTCCGCGGCCGCTTCTAGATAAGGAGGAACTACTATGGCCGGC
 
prefix:GAATTCCGCGGCCGCTTCTAGATAAGGAGGAACTACTATGGCCGGC
Line 10: Line 10:
 
suffix:ACCGGTTAATACTAGTAGCGGCCGCTGCAGT
 
suffix:ACCGGTTAATACTAGTAGCGGCCGCTGCAGT
  
 +
 +
<p align="justify">
 +
The cMyc-tag is a tag derived from the cMyc gene product. Antibodies were derived from the immunisation with synthetic peptides from the cMyc sequence [http://mcb.asm.org/content/5/12/3610.short Mol. Cell. Biol. 5,3610-3616]). The aminoacid sequence is <b>EQKLISEEDL</b>.</p>
  
 
This is a part created by the LMU-Munich 2012 team.  We added five tags to the registry, all in the Freiburg standard for N-and C-terminal fusions:
 
This is a part created by the LMU-Munich 2012 team.  We added five tags to the registry, all in the Freiburg standard for N-and C-terminal fusions:
  
[[Part:BBa_K823034|3x Flag tag],[Part:BBa_K823035|HA-tag]]
+
*[[Part:BBa_K823034|3x Flag - tag]]
 +
 
 +
*[[Part:BBa_K823035|HA - tag]]
 +
 
 +
*[[Part:BBa_K823036|cMyc - tag]]
 +
 
 +
*[[Part:BBa_K823037|His - tag]]
 +
 
 +
*[[Part:BBa_K823038| Strep - tag]]
 +
 
 +
 
 +
 
 +
 
 +
Visit our project page for more usefull parts of our [http://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks '''''BacillusB'''''io'''B'''rick'''B'''ox]. This part was also evaluated in the publication [http://www.jbioleng.org/content/7/1/29 The ''Bacillus'' BioBrick Box: generation and evaluation of essential genetic building blocks for standardized work with ''Bacillus subtilis''] by Radeck ''et al.''.
 +
 
 +
===Evaluation===
 +
<p align="justify">
 +
All 5 epitope tags were fused C- and N-terminally to GFP using the NgoMIV and AgeI restriction sites. These constructs were expressed in ''Bacillus subtils'' using [https://parts.igem.org/Part:BBa_K823026 pSB<sub>Bs</sub>0K-P<sub>spac</sub>]. This vector did not need to be induced by IPTG due to a premature stop codon in the lacI gene.
 +
 
 +
 
 +
</p>
 +
 
 +
{| style="color:black;" cellpadding="3" width="70%" cellspacing="0" border="0" align="center" style="text-align:left;"
 +
| style="width: 70%;background-color: #EBFCE4;" |
 +
{|
 +
|[[Image:LMU-Western_Blot_Tags.png|400px|center]]
 +
|-
 +
| style="width: 70%;background-color: #EBFCE4;" |
 +
{| style="color:black;" cellpadding="0" width="100%" cellspacing="0" border="0" align="center" style="text-align:center;"
 +
|style="width: 70%;background-color: #EBFCE4;" |
 +
<font color="#000000"; size="2"><p align="justify"> Fig. 1: Western blots of N- and C-terminal fusions of each tag to GFP, using the strains TMB1920 (Flag-gfp), TMB1921 (gfp-Flag), TMB1922 (HA-gfp), TMB1923 (gfp-HA), TMB1924 (cMyc-gfp), TMB1925 (gfp-cMyc), TMB1926 (His-gfp), TMB1927 (gfp-His), TMB1928 (StrepII-gfp) and TMB1929 (gfp-StrepII). For each construct, two independent clones were tested with epitope tag- and GFP-specific antibodies as a positive control.
 +
 
 +
|}
 +
|}
 +
|}
 +
 
 +
===Methods===
 +
 
 +
 
 +
To verify the functionality of the epitope tags, Western blot analyses of the strains TMB1920-TMB1929 were performed. LB medium (15 ml) was inoculated 1:100 from overnight culture and grown at 37°C and 200 rpm to OD600 ~ 0.5. Of this, 10 ml were harvested by centrifugation (8000 × g, 5 min) and the pellets stored at -20°C. Pellets were resuspended in 1 ml disruption buffer (50 mM Tris–HCl pH 7.5, 100 mM NaCl) and lysed by sonication. Samples (12 μl of lysate) were loaded per lane on two 12.5% SDS-polyacrylamide gels and SDS-PAGE was performed according standard procedure [60]. One gel was stained with colloidal coomassie, the other one was used for protein transfer to a PVDF membrane (Merck Millipore, Billerica, MA, USA) by submerged blotting procedure (Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad, Hercules, CA, USA)). After protein transfer, the membranes were treated with the following antibodies and conditions. Detailed protocols can be found [http://www.jbioleng.org/content/7/1/29/suppl/S3 here].
 +
 
 +
 
 +
''GFP''
 +
 
 +
Probing with primary antibodies takes place with rabbit anti-GFP antibodies (1:3000, Epitomics, No. 1533). Horseradish-peroxidase (HRP)-conjugated anti-rabbit antibodies (1:2000, Promega, W401B) were used as secondary antibody. Hybridization of both antibodies was carried out in Blotto-buffer (2.5% (w/v) skim milk powder, 1 × TBS (50 mM Tris–HCl pH 7.6, 0.15 M NaCl)).
 +
 
 +
 
 +
''cMyc''
 +
 
 +
Rabbit anti-Myc (1:2000, Abcan, ab9106) in TBS, 0.05% (w/v) Tween20, 5% (w/v) skim milk powder and anti-rabbit-HRP (1:2000, Promega, W401B) in Blotto-buffer were used.
 +
 
 +
Chemiluminescence signals were detected after addition of the HRP-substrate Ace Glow (Peqlab, Erlangen, Germany) using a Fusion<sup>TM</sup> imaging system (Peqlab). 
 +
</p>
 +
<br>
 +
<br>
  
  
  
,[Part:BBa_K823036|cMyc-tag],[Part:BBa_K823037|3x Flag tag],[Part:BBa_K823038|3x Flag tag]
+
==Using the myc tag with mammalian cells==
 +
We utilized this part in the composite part [https://parts.igem.org/Part:BBa_K4359006 (BBa_K4359006)]. Given that the sequence of the myc-tag is derived from the sequence of c-myc, we hypothesized that antibodies against the myc-tag may cross-react with c-myc or other components of mammalian cell lines. We tested this hypothesis by probing the whole cell lysate of SK-BR-3, a breast cancer cell line. While we were unable to ascertain whether the signal detected was from c-myc, we concluded that the anti-myc tag antibody cross-reacted with some other component(s) of the cell lysate.
  
 +
[[File:Skbr3blot.png|100px|thumb|center| Western blot of SK-BR-3 cell lysate. The membrane was probed with monoclonal anti-myc tag antibody. C-myc is 49 kDa but is known to exhibit aberrant migration in gel electrophoresis.
 +
]]
  
See here for other usefull parts of the [http://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks '''''BacillusB'''''io'''B'''rick'''B'''ox].
+
We do not recommend usage with mammalian systems.
  
<!-- Add more about the biology of this part here
+
== Team HKUST 2022 ==
===Usage and Biology===
+
Team HKUST 2022 utilize this part as a backup for protein verification.
  
 
<!-- -->
 
<!-- -->

Latest revision as of 14:28, 12 October 2022

cMyc-tag (Freiburg standard+RBS)

cMyc-tag with RBS in Freiburg standard.

Find out more about the design of our prefix with ribosome binding site.

prefix:GAATTCCGCGGCCGCTTCTAGATAAGGAGGAACTACTATGGCCGGC

suffix:ACCGGTTAATACTAGTAGCGGCCGCTGCAGT


The cMyc-tag is a tag derived from the cMyc gene product. Antibodies were derived from the immunisation with synthetic peptides from the cMyc sequence [http://mcb.asm.org/content/5/12/3610.short Mol. Cell. Biol. 5,3610-3616]). The aminoacid sequence is EQKLISEEDL.

This is a part created by the LMU-Munich 2012 team. We added five tags to the registry, all in the Freiburg standard for N-and C-terminal fusions:

  • cMyc - tag



Visit our project page for more usefull parts of our [http://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks BacillusBioBrickBox]. This part was also evaluated in the publication [http://www.jbioleng.org/content/7/1/29 The Bacillus BioBrick Box: generation and evaluation of essential genetic building blocks for standardized work with Bacillus subtilis] by Radeck et al..

Evaluation

All 5 epitope tags were fused C- and N-terminally to GFP using the NgoMIV and AgeI restriction sites. These constructs were expressed in Bacillus subtils using pSBBs0K-Pspac. This vector did not need to be induced by IPTG due to a premature stop codon in the lacI gene.

LMU-Western Blot Tags.png

Fig. 1: Western blots of N- and C-terminal fusions of each tag to GFP, using the strains TMB1920 (Flag-gfp), TMB1921 (gfp-Flag), TMB1922 (HA-gfp), TMB1923 (gfp-HA), TMB1924 (cMyc-gfp), TMB1925 (gfp-cMyc), TMB1926 (His-gfp), TMB1927 (gfp-His), TMB1928 (StrepII-gfp) and TMB1929 (gfp-StrepII). For each construct, two independent clones were tested with epitope tag- and GFP-specific antibodies as a positive control.

Methods

To verify the functionality of the epitope tags, Western blot analyses of the strains TMB1920-TMB1929 were performed. LB medium (15 ml) was inoculated 1:100 from overnight culture and grown at 37°C and 200 rpm to OD600 ~ 0.5. Of this, 10 ml were harvested by centrifugation (8000 × g, 5 min) and the pellets stored at -20°C. Pellets were resuspended in 1 ml disruption buffer (50 mM Tris–HCl pH 7.5, 100 mM NaCl) and lysed by sonication. Samples (12 μl of lysate) were loaded per lane on two 12.5% SDS-polyacrylamide gels and SDS-PAGE was performed according standard procedure [60]. One gel was stained with colloidal coomassie, the other one was used for protein transfer to a PVDF membrane (Merck Millipore, Billerica, MA, USA) by submerged blotting procedure (Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad, Hercules, CA, USA)). After protein transfer, the membranes were treated with the following antibodies and conditions. Detailed protocols can be found [http://www.jbioleng.org/content/7/1/29/suppl/S3 here].


GFP

Probing with primary antibodies takes place with rabbit anti-GFP antibodies (1:3000, Epitomics, No. 1533). Horseradish-peroxidase (HRP)-conjugated anti-rabbit antibodies (1:2000, Promega, W401B) were used as secondary antibody. Hybridization of both antibodies was carried out in Blotto-buffer (2.5% (w/v) skim milk powder, 1 × TBS (50 mM Tris–HCl pH 7.6, 0.15 M NaCl)).


cMyc

Rabbit anti-Myc (1:2000, Abcan, ab9106) in TBS, 0.05% (w/v) Tween20, 5% (w/v) skim milk powder and anti-rabbit-HRP (1:2000, Promega, W401B) in Blotto-buffer were used.

Chemiluminescence signals were detected after addition of the HRP-substrate Ace Glow (Peqlab, Erlangen, Germany) using a FusionTM imaging system (Peqlab).




Using the myc tag with mammalian cells

We utilized this part in the composite part (BBa_K4359006). Given that the sequence of the myc-tag is derived from the sequence of c-myc, we hypothesized that antibodies against the myc-tag may cross-react with c-myc or other components of mammalian cell lines. We tested this hypothesis by probing the whole cell lysate of SK-BR-3, a breast cancer cell line. While we were unable to ascertain whether the signal detected was from c-myc, we concluded that the anti-myc tag antibody cross-reacted with some other component(s) of the cell lysate.

Western blot of SK-BR-3 cell lysate. The membrane was probed with monoclonal anti-myc tag antibody. C-myc is 49 kDa but is known to exhibit aberrant migration in gel electrophoresis.

We do not recommend usage with mammalian systems.

Team HKUST 2022

Team HKUST 2022 utilize this part as a backup for protein verification.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 24
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]