Difference between revisions of "Part:BBa K3739087"

Latest revision as of 23:04, 21 October 2021

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

Histidine ammonia-lyase with a his-tag is involved in catalyzing the reaction of changing L-histidine into trans-urocanate. 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. We used his-hutH-FT to verify the secretion effect.

Usage

We ligased the induced promoter+RBS (BBa_K525998) and the parts (BBa_K3739086) on the expression vector pSB1C3 by standard assembly (BBa_K3739087). Then the ligation mixture was transformed into E.coli BL21 (DE3), and the correct recombinant one was confirmed by chloramphenicol, colony PCR and sequencing.

Characterization

1.Agarose Gel Electrophoresis
After BBa_K3739087 was constructed on vector pSB1C3 and transformed into E. coli BL21 (DE3), colony PCR was done to verify that the plasmid was correct. Data is shown below:

Fig. 1. Colony PCR results of BBa_K3739087
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.

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 200
Illegal NgoMIV site found at 636
Illegal NgoMIV site found at 1371
Illegal NgoMIV site found at 1607
Illegal AgeI site found at 273
Illegal AgeI site found at 1100
Illegal AgeI site found at 1596
1000
COMPATIBLE WITH RFC[1000]

@@ Line 5: / Line 5: @@
 Histidine ammonia-lyase with a his-tag is involved in catalyzing the reaction of changing L-histidine into trans-urocanate. 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
+===Biology===
-===Usage and Biology===
+FlAsH system was previously reported as a fluorescence detector for secreted proteins<sup>1</sup>. 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. We used his-hutH-FT to verify the secretion effect.
+===Usage===
+We ligased the induced promoter+RBS (<partinfo>BBa_K525998</partinfo>) and the parts (<partinfo>BBa_K3739086</partinfo>) on the expression vector pSB1C3 by standard assembly (<partinfo>BBa_K3739087</partinfo>). Then the ligation mixture was transformed into ''E.coli'' BL21 (DE3), and the correct recombinant one was confirmed by chloramphenicol, colony PCR and sequencing.
+===Characterization===
+.Agarose Gel Electrophoresis<br/>
+After <partinfo>BBa_K3739087</partinfo> was constructed on vector pSB1C3 and transformed into ''E. coli'' BL21 (DE3), colony PCR was done to verify that the plasmid was correct. Data is shown below:<br/>
+[[File:T--XMU-China--86-1.png|300px]]<br/>
+'''Fig. 1.''' Colony PCR results of BBa_K3739087<br/>
+. Qualification of LMT secretion efficiency<br/>
+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 OD<sub>600</sub>.<br/>
+[[File:T--XMU-China--86-2.png|300px]]<br/>
+'''Fig. 2.''' Fluorescence intensity/OD<sub>600</sub> of cell supernatant after incubation in the dark for 1h.<br/>
+The result shows that the fluorescence intensity/OD<sub>600</sub> of the group Aly01 and LMT is significantly higher than native control group, which proves that these 2 signal peptides can function well.
+===Reference===
+.	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.
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