Difference between revisions of "Part:BBa K3739084"
Line 5: Line 5:
 
Histidine ammonia-lyase with a his-tag is involved in catalyzing the reaction of changing L-histidine into trans-urocanate. Aly01 is a signal peptile. The proteins labeled of tetracysteine motif tag (FlAsH tag, FT in short) can be detected with the biarsenical compound FlAsH.  
 
Histidine ammonia-lyase with a his-tag is involved in catalyzing the reaction of changing L-histidine into trans-urocanate. Aly01 is a signal peptile. The proteins labeled of tetracysteine motif tag (FlAsH tag, FT in short) can be detected with the biarsenical compound FlAsH.  
  
−
<!-- Add more about the biology of this part here
+
 
−
===Usage and Biology===
+
=== Biology===
 +
FlAsH system was previously reported as a fluorescence detector for secreted proteins1. The protein labeled of tetracysteine motif tag (FlAsH tag, FT in short) can react with biarsenical compound FlAsH, showing great fluorescence at 528 nm emission. FlAsH system is used to quantify and qualify the secretion efficiency. Aly01 is a signal peptide, and we used Aly01-his-hutH-FT to verify the secretion effect of Aly01.
 +
 
 +
=== Usage===
 +
We ligased the induced promoter+RBS (<partinfo>BBa_K525998</partinfo>) and the parts (<partinfo>BBa_K3739082</partinfo>) on the expression vector pET-28a(+) by standard assembly (<partinfo>BBa_K3739084</partinfo>). Then the ligation mixture was transformed into E.coli BL21 (DE3), and the correct recombinant one was confirmed by kanamycin, colony PCR and sequencing.
 +
 
 +
=== Characterization===
 +
====1. Agarose Gel Electrophoresis====
 +
After <partinfo>BBa_K3739084</partinfo> was constructed on vector pET-28a(+) and transformed into ''E.coli'' BL21 (DE3), colony PCR was done to verify that the plasmid was correct. Data is shown below:
 +
 
 +
[[File:T--XMU-China--K3739084.png|500px]]
 +
 
 +
'''Fig. 1.''' Colony PCR results of BBa_K3739084
 +
 
 +
====2. Qualification of LMT secretion efficiency====
 +
 
 +
After cultivation for 5 hours at 37℃ and induction for 2 hours with 0.1mM IPTG, the cell supernatant was separated by centrifugation. The supernatant along with 2 μM FlAsH-EDT2 and 1 mM DTT were added to wells in a 96-well plate. Following incubation in the dark for 1 h at 37 °C, fluorescence was measured by 503 nm excitation and 528 nm emission, and each value was normalized by OD600.
 +
 
 +
[[File:T--XMU-China--FT_Qualify.png|300px]]
 +
 
 +
'''Fig. 2.''' Fluorescence intensity/OD600 of cell supernatant after incubation in the dark for 1h.
 +
 
 +
The result shows that the fluorescence intensity/OD600 of the group Aly01 and LMT is significantly higher than native control group, which proves that these 2 signal peptides can function well.
 +
 
 +
===Reference===
 +
1. Haitjema, C. H.; Boock, J. T.; Natarajan, A.; Dominguez, M. A.; Gardner, J. G.; Keating, D. H.; Withers, S. T.; DeLisa, M. P., Universal Genetic Assay for Engineering Extracellular Protein Expression. '''ACS Synthetic Biology''' 2014, 3 (2), 74-82.
 +
 
  
 
<!-- -->
 
<!-- -->

Revision as of 19:43, 21 October 2021


K525998(T7-RBS)-Aly01-his-hutH-FT

Histidine ammonia-lyase with a his-tag is involved in catalyzing the reaction of changing L-histidine into trans-urocanate. Aly01 is a signal peptile. The proteins labeled of tetracysteine motif tag (FlAsH tag, FT in short) can be detected with the biarsenical compound FlAsH.


Biology

FlAsH system was previously reported as a fluorescence detector for secreted proteins1. The protein labeled of tetracysteine motif tag (FlAsH tag, FT in short) can react with biarsenical compound FlAsH, showing great fluorescence at 528 nm emission. FlAsH system is used to quantify and qualify the secretion efficiency. Aly01 is a signal peptide, and we used Aly01-his-hutH-FT to verify the secretion effect of Aly01.

Usage

We ligased the induced promoter+RBS (BBa_K525998) and the parts (BBa_K3739082) on the expression vector pET-28a(+) by standard assembly (BBa_K3739084). Then the ligation mixture was transformed into E.coli BL21 (DE3), and the correct recombinant one was confirmed by kanamycin, colony PCR and sequencing.

Characterization

1. Agarose Gel Electrophoresis

After BBa_K3739084 was constructed on vector pET-28a(+) and transformed into E.coli BL21 (DE3), colony PCR was done to verify that the plasmid was correct. Data is shown below:

T--XMU-China--K3739084.png

Fig. 1. Colony PCR results of BBa_K3739084

2. Qualification of LMT secretion efficiency

After cultivation for 5 hours at 37℃ and induction for 2 hours with 0.1mM IPTG, the cell supernatant was separated by centrifugation. The supernatant along with 2 μM FlAsH-EDT2 and 1 mM DTT were added to wells in a 96-well plate. Following incubation in the dark for 1 h at 37 °C, fluorescence was measured by 503 nm excitation and 528 nm emission, and each value was normalized by OD600.

T--XMU-China--FT Qualify.png

Fig. 2. Fluorescence intensity/OD600 of cell supernatant after incubation in the dark for 1h.

The result shows that the fluorescence intensity/OD600 of the group Aly01 and LMT is significantly higher than native control group, which proves that these 2 signal peptides can function well.

Reference

1. Haitjema, C. H.; Boock, J. T.; Natarajan, A.; Dominguez, M. A.; Gardner, J. G.; Keating, D. H.; Withers, S. T.; DeLisa, M. P., Universal Genetic Assay for Engineering Extracellular Protein Expression. ACS Synthetic Biology 2014, 3 (2), 74-82.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 275
    Illegal NgoMIV site found at 711
    Illegal NgoMIV site found at 1446
    Illegal NgoMIV site found at 1682
    Illegal AgeI site found at 111
    Illegal AgeI site found at 348
    Illegal AgeI site found at 1175
    Illegal AgeI site found at 1671
  • 1000
    COMPATIBLE WITH RFC[1000]