Difference between revisions of "Part:BBa K3815007"
Line 2: Line 2:
 
__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K3815007 short</partinfo>
 
<partinfo>BBa_K3815007 short</partinfo>
−
<h3><font size="4.5">Descriotion of this part</font> </h3>
+
<h3><font size="4.5">Description of this part</font> </h3>
 
<h3><font size="3">Targeted protein</font> </h3>
 
<h3><font size="3">Targeted protein</font> </h3>
 
This part is for the purfication of antimicrobial peptide,Defensin1. This is derived from <i>Homo sapiens</i>. This can inhibit the growth of the gram-positive bacteria. In our experiment, we used it to kill the bacteria in the vase water.<br><br>
 
This part is for the purfication of antimicrobial peptide,Defensin1. This is derived from <i>Homo sapiens</i>. This can inhibit the growth of the gram-positive bacteria. In our experiment, we used it to kill the bacteria in the vase water.<br><br>
 
<h3><font size="3">Purification system</font> </h3>
 
<h3><font size="3">Purification system</font> </h3>
−
This part is composed of His tag, Mxe Gyr intein, PT linker, and targeted protein. This is for the peptide intein tag purification.  Producing peptide with His tag, it is recovered by Ni chromatography. After that, adding DTT to it, the N terminal of Mxe GyrA intein is cut. Then, we get the targeted protein.
+
This part is composed of His tag, Mxe Gyr intein, PT linker, and targeted protein. This is for the peptide intein tag purification.  Producing peptide with His tag, it is recovered by Ni chromatography. After that, adding DTT to it, the N terminal of Mxe GyrA intein is cut. Then, we get the targeted protein. This method saves the time removing the tags compared to the method using only tags without intein.
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
Line 17: Line 17:
 
<ul>
 
<ul>
 
<li>Cells were grown in 1000ml LB media at 37<sup>o</sup>C shaking at 180 rpm.
 
<li>Cells were grown in 1000ml LB media at 37<sup>o</sup>C shaking at 180 rpm.
−
<li>when the OD exceeded 0.35, 1 M IPTG 2ml was added to induce the peptide expression.
+
<li>when the OD exceeded 0.35, 1 M IPTG 200μL was added to induce the peptide expression.
 
<li>Incubate at 30℃ at 180rpm for 6 hours after adding IPTG.
 
<li>Incubate at 30℃ at 180rpm for 6 hours after adding IPTG.
 
</ul>
 
</ul>
Line 27: Line 27:
 
<br>
 
<br>
 
Fig1 shows the result of SDS-PAGE.
 
Fig1 shows the result of SDS-PAGE.
−
The lane 4, 8,and 12 are the result of NOP1.<br>  NOP1 is 1132Da, so these date shows that we could not confirm its production.
+
The lane 2 and 8 are the result of Defensin1.<br>  Defensin1 is 5713Da, so these date shows that we could not confirm its production.
  
  

Revision as of 12:19, 20 October 2021


Defensin1-Mxe GryA intein-PT-linker-His tag

Description of this part

Targeted protein

This part is for the purfication of antimicrobial peptide,Defensin1. This is derived from Homo sapiens. This can inhibit the growth of the gram-positive bacteria. In our experiment, we used it to kill the bacteria in the vase water.

Purification system

This part is composed of His tag, Mxe Gyr intein, PT linker, and targeted protein. This is for the peptide intein tag purification. Producing peptide with His tag, it is recovered by Ni chromatography. After that, adding DTT to it, the N terminal of Mxe GyrA intein is cut. Then, we get the targeted protein. This method saves the time removing the tags compared to the method using only tags without intein.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 249
    Illegal SpeI site found at 573
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 249
    Illegal SpeI site found at 573
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 249
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 249
    Illegal SpeI site found at 573
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 249
    Illegal SpeI site found at 573
    Illegal NgoMIV site found at 682
  • 1000
    COMPATIBLE WITH RFC[1000]

Expression

  • Cells were grown in 1000ml LB media at 37oC shaking at 180 rpm.
  • when the OD exceeded 0.35, 1 M IPTG 200μL was added to induce the peptide expression.
  • Incubate at 30℃ at 180rpm for 6 hours after adding IPTG.

Purification

1.When this fused protein were produced, it was recovered by Ni chromatography
2.Adding DTT to it, the targeted protein was cut out by the cleavage of intein.
3.SDSPAGE was performed in order to confirm the presence of it.

Fig1 shows the result of SDS-PAGE. The lane 2 and 8 are the result of Defensin1.
Defensin1 is 5713Da, so these date shows that we could not confirm its production.