Difference between revisions of "Part:BBa K2321010"

 
 
(13 intermediate revisions by one other user not shown)
Line 12: Line 12:
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2321010 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2321010 SequenceAndFeatures</partinfo>
−
 
+
<br/>
−
 
+
<br/>
 +
=Improvement by ICJFLS2022=
 +
==Overview:==
 +
<br/>
 +
AmilCP serving as a reporter protein exhibits strong color when it is expressed. It is visible to naked eyes, thereby requiring no instruments to observe, making it to be a useful reporter protein. In our project this year, toehold switch sequence was linked to the upstream of AmilCP to construct the recombined plasmid pET-28a-toehold switch-AmilCP. It can express AmilCP protein triggered by miRNA 34a-5p, which is used to detect the amount of miRNA 34a-5p in samples, such as serum or blood.
 +
<br/>
 +
<br/>
 +
==Results:==
 +
<br/>
 +
First, pSB1C3-AmilCP was transfected into BL21 bacteria to confirm the expression of AmilCP, which is shown in Fig.1.
 +
<br/>
 +
<br/>
 +
[[File:K2321010-3.jpg|center]]
 +
Fig.1 The clones containing plasmid pSB1C3-amilCP cultured on the agar plate expressed AmilCP protein, exhibiting strong blue/purple color.
 +
<br/>
 +
<br/>
 +
<br/>
 +
To construct the standard part, toehold switch-amilCP was synthesized and checked the restriction enzyme information, which is shown as follows:
 +
<br/>
 +
<br/>
 +
[[File:K4167000-fig.1-2.jpg|center]]
 +
Fig.2 The map of toehold switch-amilCP described with SnapGene Viewer, showing the restriction enzyme information (no EcoRI and PstI sites).
 +
<br/>
 +
<br/>
 +
<br/>
 +
After detecting the restriction enzyme information of toehold switch-amilCP using SnapGene software, it was inserted into the pSB1C3 plasmid to construct the standard part pSB1C3-toehold switch-amilCP with PCR method. Then it was identified as follows:
 +
<br/>
 +
<br/>
 +
[[File:K4167000-fig.2.jpg|center]]
 +
Fig.3 Identification of standard part pSB1C3-toehold switch-amilCP using PCR and digestion with EcoRI and PstI. 
 +
M: Marker; 1: PCR result; Digestion result. 
 +
<br/>
 +
<br/>
 +
Toehold switch-amilCP plasmid is designed to express the amilCP protein controlled by the toehold switch and miRNA 34a-5p. It comprises the antisense sequence of miRNA 34a-5p, RBS, Linker and part sequence of miRNA 34a-5p, which form a toehold switch, as well as the gene of marker protein amilCP. At the presence of miRNA 34a-5p, it binds to its antisense sequence, opening the toehold switch to trigger the expression of amilCP, which is easily measured. The mechanism is shown as Fig.4.
 +
<br/>
 +
<br/>
 +
[[File:K4167000-fig.3-2.jpg|center]]
 +
Fig.4 The mechanism of toehold switch-amilCP.
 +
<br/>
 +
<br/>
 +
<br/>
 +
Toehold switch-amilCP was also cloned into pET-28a expression vector, constructing the recombined plasmid pET-28a-toehold switch-amilCP. After it was transfected into BL21 strain, no amilCP protein (purple color) could be observed with naked eyes, indicating that the toehold switch was effective. However, after transfection with miRNA 34a-5p into the BL21 strain transfected with pET-28a-toehold switch-amilCP, some transfected clones appeared purple color, which were shown in Fig.5.
 +
<br/>
 +
<br/>
 +
[[File:K4167000-fig.4.jpg|center]]
 +
Fig.5 The effectiveness of toehold switch-amilCP.
 +
Bacteria clones only transfected with toehold switch-amilCP appeared white color, while bacteria clones transfected with both toehold switch-amilCP and miRNA 34a-5p appeared purple color (miRNA 34a-5p switched on the expression of amilCP).
 +
<br/>
 +
<br/>
 +
<br/>
 +
To increase the yielding of marker protein amilCP, some different culture conditions were optimized, including the pH value, temperature, fermentation time, and the concentration of transfected miRNA. BL21 strain containing toehold switch plasmid were cultured under different conditions. Since reporter protein amilCP has color, we can easily intuitively find the optimal conditions through the change of color. The optimization experiment results indicated that pH7.2, 37°C, fermentation 18h, and 1.5uM miRNA are the best culture conditions for higher reporter protein production in E. coli.
 +
<br/>
 +
<br/>
 +
[[File:K4167000-fig.5-2.jpg|center]]
 +
Fig.6 Optimization of culture conditions of BL21 strain with toehold switch-amilCP plasmid and miRNA 34a-5p.
 +
<br/>
 +
<br/>
 +
<br/>
 +
=References=
 +
1. Wan Y, Liu Y, Wang X, Wu J, Liu K, Zhou J, Liu L, Zhang C. Identification of differential microRNAs in cerebrospinal fluid and serum of patients with major depressive disorder. PLoS One, 2015 Mar 12;10(3): e0121975. doi: 10.1371/journal.pone.0121975
 +
<br/>
 +
2. Zhou L, Zhu Y, Chen W, Tang Y. Emerging role of microRNAs in major depressive disorder and its implication on diagnosis and therapeutic response.  J Affect Disord. 2021 May 1;286: 80-86. doi: 10.1016/j.jad.2021.02.063
 +
<br/>
 +
3. Green AA, Silver PA, Collins JJ, Yin P. Toehold switches: de-novo-designed regulators of gene expression. Cell. 2014 Nov 6;159(4):925-39. doi: 10.1016/j.cell.2014.10.002
 +
<br/>
 +
<br/>
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K2321010 parameters</partinfo>
 
<partinfo>BBa_K2321010 parameters</partinfo>
 
<!-- -->
 
<!-- -->

Latest revision as of 06:37, 10 October 2022


amilCP, blue chromoprotein expression device


This chromoprotein is from the coral Acropora millepora. amilCP naturally exhibits strong color when it is expressed. The protein has an absorbance maximum at 588 nm giving it a blue/purple color visible to the naked eye, thereby requiring no instruments to observe. The strong color is readily observed in both LB or agar culture, in less than 24 hours of incubation.

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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]



Improvement by ICJFLS2022

Overview:


AmilCP serving as a reporter protein exhibits strong color when it is expressed. It is visible to naked eyes, thereby requiring no instruments to observe, making it to be a useful reporter protein. In our project this year, toehold switch sequence was linked to the upstream of AmilCP to construct the recombined plasmid pET-28a-toehold switch-AmilCP. It can express AmilCP protein triggered by miRNA 34a-5p, which is used to detect the amount of miRNA 34a-5p in samples, such as serum or blood.

Results:


First, pSB1C3-AmilCP was transfected into BL21 bacteria to confirm the expression of AmilCP, which is shown in Fig.1.

K2321010-3.jpg

Fig.1 The clones containing plasmid pSB1C3-amilCP cultured on the agar plate expressed AmilCP protein, exhibiting strong blue/purple color.


To construct the standard part, toehold switch-amilCP was synthesized and checked the restriction enzyme information, which is shown as follows:

K4167000-fig.1-2.jpg

Fig.2 The map of toehold switch-amilCP described with SnapGene Viewer, showing the restriction enzyme information (no EcoRI and PstI sites).


After detecting the restriction enzyme information of toehold switch-amilCP using SnapGene software, it was inserted into the pSB1C3 plasmid to construct the standard part pSB1C3-toehold switch-amilCP with PCR method. Then it was identified as follows:

K4167000-fig.2.jpg

Fig.3 Identification of standard part pSB1C3-toehold switch-amilCP using PCR and digestion with EcoRI and PstI. M: Marker; 1: PCR result; Digestion result.

Toehold switch-amilCP plasmid is designed to express the amilCP protein controlled by the toehold switch and miRNA 34a-5p. It comprises the antisense sequence of miRNA 34a-5p, RBS, Linker and part sequence of miRNA 34a-5p, which form a toehold switch, as well as the gene of marker protein amilCP. At the presence of miRNA 34a-5p, it binds to its antisense sequence, opening the toehold switch to trigger the expression of amilCP, which is easily measured. The mechanism is shown as Fig.4.

K4167000-fig.3-2.jpg

Fig.4 The mechanism of toehold switch-amilCP.


Toehold switch-amilCP was also cloned into pET-28a expression vector, constructing the recombined plasmid pET-28a-toehold switch-amilCP. After it was transfected into BL21 strain, no amilCP protein (purple color) could be observed with naked eyes, indicating that the toehold switch was effective. However, after transfection with miRNA 34a-5p into the BL21 strain transfected with pET-28a-toehold switch-amilCP, some transfected clones appeared purple color, which were shown in Fig.5.

K4167000-fig.4.jpg

Fig.5 The effectiveness of toehold switch-amilCP. Bacteria clones only transfected with toehold switch-amilCP appeared white color, while bacteria clones transfected with both toehold switch-amilCP and miRNA 34a-5p appeared purple color (miRNA 34a-5p switched on the expression of amilCP).


To increase the yielding of marker protein amilCP, some different culture conditions were optimized, including the pH value, temperature, fermentation time, and the concentration of transfected miRNA. BL21 strain containing toehold switch plasmid were cultured under different conditions. Since reporter protein amilCP has color, we can easily intuitively find the optimal conditions through the change of color. The optimization experiment results indicated that pH7.2, 37°C, fermentation 18h, and 1.5uM miRNA are the best culture conditions for higher reporter protein production in E. coli.

K4167000-fig.5-2.jpg

Fig.6 Optimization of culture conditions of BL21 strain with toehold switch-amilCP plasmid and miRNA 34a-5p.


References

1. Wan Y, Liu Y, Wang X, Wu J, Liu K, Zhou J, Liu L, Zhang C. Identification of differential microRNAs in cerebrospinal fluid and serum of patients with major depressive disorder. PLoS One, 2015 Mar 12;10(3): e0121975. doi: 10.1371/journal.pone.0121975
2. Zhou L, Zhu Y, Chen W, Tang Y. Emerging role of microRNAs in major depressive disorder and its implication on diagnosis and therapeutic response. J Affect Disord. 2021 May 1;286: 80-86. doi: 10.1016/j.jad.2021.02.063
3. Green AA, Silver PA, Collins JJ, Yin P. Toehold switches: de-novo-designed regulators of gene expression. Cell. 2014 Nov 6;159(4):925-39. doi: 10.1016/j.cell.2014.10.002