Difference between revisions of "Part:BBa K2262005"
 
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<h1>'''Introduction'''</h1>
 
<h1>'''Introduction'''</h1>
 +
 
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
−
This peptide possesses antifungal activity sensitive to inorganic cations from the defensin family and was proved to have an anti-fungal function. NCTU_Formosa used it to be one of a training data for creating our anti-fungal peptide scoring card.
+
This peptide possesses antifungal activity sensitive to inorganic cations from the defensin family and was proved to have an anti-fungal function [1]. NCTU_Formosa used it to be one of a training data for forming the antifungal peptide scoring card.
  
 
<br>
 
<br>
  
 
<h1>'''Experiment'''</h1>
 
<h1>'''Experiment'''</h1>
 +
<br>
 +
 
<p style="padding:1px;font-size:16px"><b>1. Cloning </b></p>
 
<p style="padding:1px;font-size:16px"><b>1. Cloning </b></p>
 
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
−
We put T7 promoter, RBS, and Sa-AFP1 together with pSB1C3 as a backbone. Then we conducted Taq PCR to check the size of the DNA sequence was right. The length of T7 promoter+RBS+Sa-AFP1 is around 250 ~ 500 b.p.. Its PCR product is around 500 ~ 750 b.p..
+
We put T7 promoter, RBS, and Sa-AFP1 together with pSB1C3 as a backbone. Then we conducted Taq PCR to check the size of the DNA sequence was right. The length of T7 promoter+RBS+Sa-AFP1 is234 b.p. Its PCR product is around 546 b.p.
  
 
<br>
 
<br>
 
[[File:Agarose gel Sa-AFP1.png|200px|thumb|center|'''Figure 2.''' P<sub>T7</sub> + RBS + Sa-AFP1 <br>
 
[[File:Agarose gel Sa-AFP1.png|200px|thumb|center|'''Figure 2.''' P<sub>T7</sub> + RBS + Sa-AFP1 <br>
−
The DNA sequence length of P<sub>T7</sub> + RBS + SSa-AFP1 is around 200 ~ 250 b.p.. The size of its PCR product should be close to 500 ~ 550 b.p..]]
+
The DNA sequence length of P<sub>T7</sub> + RBS + SSa-AFP1 is around 200 ~ 250 b.p. The size of its PCR product should be close to 500 ~ 550 b.p.]]
  
 
<br>
 
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<p style="padding:1px;font-size:16px"><b>2. Expressing</b></p>
 
<p style="padding:1px;font-size:16px"><b>2. Expressing</b></p>
 
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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Because Sa-AFP1 had not been expressed by E. coli before, it was a new challenge.The strain we chose to make Sa-AFP1 was E.coli Rosetta-gami strain, which can form the disulfide bonds in the cytoplasm to express the protein.To ensure the peptide was produced, after breaking the bacteria and boiling the lysate with sample buffer and β-mercaptoethanol mixture for 15 minutes, we run SDS-PAGE to check the mass of the peptide. The mass of Sa-AFP1 is around 5.6 kDa.
+
Because Sa-AFP1 had not been expressed by <i>E. coli</i> before, it was a new challenge. The strain we chose to make Sa-AFP1 was <i>E. coli</i> Rosetta-gami strain, which forms the disulfide bonds in the cytoplasm to express the protein. To ensure the peptide was produced, after breaking the bacteria and boiling the lysate with sample buffer and β-mercaptoethanol mixture for 15 minutes, we run SDS-PAGE to check the mass of the peptide. The mass of Sa-AFP1 is around 5.7 kDa.
 +
<br>
 +
<br>
 +
[[File:SDS Sa-AFP.png|500px|thumb|center|'''Figure 3.'''The mass of Sa-AFP1 is around 5.7 kDa. The result shows that NO.2, No.3, NO.4, No.5, and No.6 had made the peptide.]]
  
 
<br>
 
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<h1>'''Safety'''</h1>
 
<h1>'''Safety'''</h1>
 
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
−
To make sure the lysate will not contain any alive bacteria, we boiled the lysate for 15 minutes after breaking the bacteria. The result of SDS-PAGE shows the peptide still exist after boiling it. So the only thing we need to know was that whether the lysate would have alive bacteria after boiling. We put boiled lysate on an LB plate and cultured it at 37℃ for 16 hours. The result shows that there are no alive E. coli after boiling for 15 minutes.  
+
To make sure the lysate will not contain any living bacteria, we boiled the lysate for 15 minutes after sonicating the bacteria. The result of SDS-PAGE shows that the peptide still exists after boiling it. So, the only thing we need to know was that whether the lysate would have living bacteria after boiling. We put boiled lysate on an LB plate and cultured it at 37℃ for 16 hours. The result shows that there is no living <i>E. coli</i> after boiling it for 15 minutes.
  
 
<br>
 
<br>
 
   
 
   
−
[[File:NCTU Formosa 2017 safety.png|400px|thumb|center|'''Figure 2.''' The LB plate is divided into 8 parts. Each of parts is different time interval boiling in water. The control group is only LB solution. The other 7 ones are boiling for 0, 5, 10, 15, 20, 25, 30 minutes separately.  ]]
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[[File:NCTU Formosa 2017 safety.png|400px|thumb|center|'''Figure 4.''' The LB plate is divided into 8 parts. Each of parts is different time interval boiling in water. The control group is only LB solution. The other 7 ones are boiling for 0, 5, 10, 15, 20, 25, 30 minutes separately.  ]]
  
 
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<h1>'''Reference'''</h1>
 
<h1>'''Reference'''</h1>
  
−
Terras FR1; Torrekens S; Van Leuven F; Osborn RW; Vanderleyden J; Cammue BP; Broekaert WF. "A new family of basic cysteine-rich plant antifungal proteins from Brassicaceae species." FEBS Lett. 1993 Feb 1;316(3):233-40.
+
[1] Terras FR1; Torrekens S; Van Leuven F; Osborn RW; Vanderleyden J; Cammue BP; Broekaert WF. "A new family of basic cysteine-rich plant antifungal proteins from Brassicaceae species." FEBS Lett. 1993 Feb 1;316(3):233-40.
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 15:13, 7 December 2017


T7 Promoter+RBS+Sa-AFP1



Figure 1. PT7+RBS+Sa-AFP1+terminator



Introduction

      This peptide possesses antifungal activity sensitive to inorganic cations from the defensin family and was proved to have an anti-fungal function [1]. NCTU_Formosa used it to be one of a training data for forming the antifungal peptide scoring card.


Experiment


1. Cloning

      We put T7 promoter, RBS, and Sa-AFP1 together with pSB1C3 as a backbone. Then we conducted Taq PCR to check the size of the DNA sequence was right. The length of T7 promoter+RBS+Sa-AFP1 is234 b.p. Its PCR product is around 546 b.p.


Figure 2. PT7 + RBS + Sa-AFP1
The DNA sequence length of PT7 + RBS + SSa-AFP1 is around 200 ~ 250 b.p. The size of its PCR product should be close to 500 ~ 550 b.p.


2. Expressing

      Because Sa-AFP1 had not been expressed by E. coli before, it was a new challenge. The strain we chose to make Sa-AFP1 was E. coli Rosetta-gami strain, which forms the disulfide bonds in the cytoplasm to express the protein. To ensure the peptide was produced, after breaking the bacteria and boiling the lysate with sample buffer and β-mercaptoethanol mixture for 15 minutes, we run SDS-PAGE to check the mass of the peptide. The mass of Sa-AFP1 is around 5.7 kDa.

Figure 3.The mass of Sa-AFP1 is around 5.7 kDa. The result shows that NO.2, No.3, NO.4, No.5, and No.6 had made the peptide.



Safety

      To make sure the lysate will not contain any living bacteria, we boiled the lysate for 15 minutes after sonicating the bacteria. The result of SDS-PAGE shows that the peptide still exists after boiling it. So, the only thing we need to know was that whether the lysate would have living bacteria after boiling. We put boiled lysate on an LB plate and cultured it at 37℃ for 16 hours. The result shows that there is no living E. coli after boiling it for 15 minutes.


Figure 4. The LB plate is divided into 8 parts. Each of parts is different time interval boiling in water. The control group is only LB solution. The other 7 ones are boiling for 0, 5, 10, 15, 20, 25, 30 minutes separately.



Reference

[1] Terras FR1; Torrekens S; Van Leuven F; Osborn RW; Vanderleyden J; Cammue BP; Broekaert WF. "A new family of basic cysteine-rich plant antifungal proteins from Brassicaceae species." FEBS Lett. 1993 Feb 1;316(3):233-40.

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]