Difference between revisions of "Part:BBa K1694002"

 
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<h1>'''Introduction'''</h1>
 
<h1>'''Introduction'''</h1>
 
[[File:ON.png|200px|thumb|right|'''Fig.1''' Lpp-OmpA-NcoI]]  
 
[[File:ON.png|200px|thumb|right|'''Fig.1''' Lpp-OmpA-NcoI]]  
Lpp-OmpA protein is the expression system of outer membrane protein, which consists of the 20 amino acids of signal sequence, the nine N-terminal amino acids of the lipoprotein (Lpp) and the residual 46-159 amino acids of the OmpA.  
+
Lpp-OmpA protein is the expression system of outer membrane protein, which consists of the 20 amino acids of signal sequence, the 9 N-terminal amino acids of the lipoprotein (Lpp) and the residual 46-159 amino acids of the OmpA.  
 
The lipoprotein (Lpp) is the most abundant protein on the outer membrane, which possesses the function of targeting to the outer membrane.  
 
The lipoprotein (Lpp) is the most abundant protein on the outer membrane, which possesses the function of targeting to the outer membrane.  
The OmpA domain constitutes eight-stranded β-barrel to construct an anchor on the outer membrane, providing the stable expression of the scFv.
+
The OmpA domain constitutes eight-stranded β-barrel to construct an anchor on the outer membrane, providing the stable expression of the protein displayed on the outer membrane. By taking advantage of efficient targeting OmpA to the outer membrane, it allows C-terminal fusion of the protein sequence to be displayed out of the outer membrane.
By taking advantage of efficient targeting OmpA to the outer membrane, it allows C-terminal fusion of the scFv sequence to be displayed out of the outer mambrane.
+
  
 
<h1>'''Modifying and Improving the Existed Biobrick</h1>
 
<h1>'''Modifying and Improving the Existed Biobrick</h1>
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<html>
 
<html>
 
<a href="https://parts.igem.org/Part:BBa_K103006">BBa_K103006</a>
 
<a href="https://parts.igem.org/Part:BBa_K103006">BBa_K103006</a>
</html>. The most important part that we modified is that we replaced the long GS linker behide the transmembrane protein straightly to a six-amino-acid restriction site, ''Nco''I. This changing has two benefits. First, the appearance of ''Nco''I allowing the linked protein, for example in our project, scFv to be easily changed and to be brought outside conveniently (like a cassette). Furthermore, ''Nco''I also can act as a useful linker in fusion protein (between transmembrane protein and the linked protein), this has been proved in our project. Moreover, we also adjust the amino acids of OmpA from twenty-five to one hundred thirty-eight to shorten the distance between linked protein (scFv) and the outer membrane. This modified is useful for construction when less flexible of linked protein is considered.
+
</html>. The most contributed modification to this biobrick is that we replaced the long GS linker behide the transmembrane protein but we chose a six nucleotides restriction site, ''NcoI'', to make it express remarkably on the outer membrane. This change has two benefits. First, the appearance of ''NcoI'' allowed the linked protein, for example scFv in our project, to be easily changed and to be brought outside conveniently (like a cassette). Second, ''NcoI'' also can act as an useful linker in fusion protein (between transmembrane protein and the linked protein), this has been proved in our project. Moreover, we also selected the amino acids of OmpA from the forty-sixth till the one hundred fifty-ninth and the function of Lpp-OmpA to bring scFv onto the outer membrane <i>E. coli </i>is completely proved by our experiments.
  
<h2>'''The features of Lpp-OmpA expression system'''</h2>
+
<h2>'''An alternative version with BamHI site'''</h2>
<br><br>
+
If you'd like to directly use this part as a vector/backbone for cloning a fusion protein, you should be noticed that an extra NcoI site is present on Cm resistance gene. <html>
'''The special restriction site─<i>Nco</i>I:''' This design avoids the dilemma of the mixed restriction sites.<br><br>
+
<a href="https://parts.igem.org/Part:BBa_K1991004">BBa_K1991004</a>
'''The versatile applications:''' The diversity of introduced protein connecting to OmpA can provide the multiple functions outside the surface of the <i>E.coli</i>, for instance, we can change every kinds of scFv we want.<br><br>
+
</html> replaced NcoI site behind LO with BamHI site and provides an alternative choice for cloning a LO fusion protein
 +
 
 +
 
 +
<h2>'''The features of Lpp-OmpA expression system'''</h2><br>
 +
'''The versatile applications:''' The diversity of introduced protein connecting to OmpA can provide the multiple functions outside the surface of the <i>E. coli</i>. For instance, in our project, we can change every kind of scFv we desire.<br><br>
 
'''The stabilization of expression:''' Due to the anchoring capabilities of lipoprotein and outer membrane protein, the Lpp-OmpA expression system can display passenger proteins more efficiently and also performs the strong adaptability to the various passenger protens.<br><br>
 
'''The stabilization of expression:''' Due to the anchoring capabilities of lipoprotein and outer membrane protein, the Lpp-OmpA expression system can display passenger proteins more efficiently and also performs the strong adaptability to the various passenger protens.<br><br>
  
 
<h1>'''Experiment'''</h1>
 
<h1>'''Experiment'''</h1>
After receiving the DNA sequences from the gene synthesis company, we recombined each Lpp-OmpA-N gene to PSB1C3 backbones and conducted a PCR experiment to check the size of each of the Lpp-OmpA-N. The DNA sequence length of the Lpp-OmpA-N is around 400~500 bp. In this PCR experiment, the Lpp-OmpA-N products size should be near at 650~750 bp. The Fig.2 showed the correct size of the Lpp-OmpA-N, and proved that we successful ligated the Lpp-OmpA-N sequence onto an ideal backbone.
+
After receiving the DNA sequences from the gene synthesis company, we recombined each Lpp-OmpA-N gene to pSB1C3 backbones and conducted a PCR experiment to check the size of each of the Lpp-OmpA-N. The DNA sequence length of the Lpp-OmpA-N is around 400~500 bp. In this PCR experiment, the Lpp-OmpA-N products size should be near at 650~750 bp. '''Fig.2''' showed the correct size of the Lpp-OmpA-N, and proved that we successful ligated the Lpp-OmpA-N sequence onto an ideal backbone.
  
[[File:Lpp-OmpA-NcoI.png|200px|thumb|left|'''Fig.2''' The DNA sequence length of the Lpp-OmpA-N is around 400~500 bp. In this PCR experiment, the Lpp-OmpA-N products size should be near at 650~750 bp.]]
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[[File:Lpp-OmpA-NcoI.png|200px|thumb|left|'''Fig.2'''<br> A:Lpp-OmpA-NcoI The DNA sequence length of the Lpp-OmpA-N is around 400~550 bp. In this PCR experiment, the Lpp-OmpA-N products size should be close to 650~800 bp.]]
[[File:OmpA-N.png|600px|thumb|center|'''Fig.3''' Lpp-OmpA-NcoI]]  
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[[File:OmpA-N.png|600px|thumb|center|'''Fig.3''' Plate of Lpp-OmpA-NcoI]]  
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 08:50, 10 October 2016

Lpp-OmpA-N

Introduction

Fig.1 Lpp-OmpA-NcoI

Lpp-OmpA protein is the expression system of outer membrane protein, which consists of the 20 amino acids of signal sequence, the 9 N-terminal amino acids of the lipoprotein (Lpp) and the residual 46-159 amino acids of the OmpA. The lipoprotein (Lpp) is the most abundant protein on the outer membrane, which possesses the function of targeting to the outer membrane. The OmpA domain constitutes eight-stranded β-barrel to construct an anchor on the outer membrane, providing the stable expression of the protein displayed on the outer membrane. By taking advantage of efficient targeting OmpA to the outer membrane, it allows C-terminal fusion of the protein sequence to be displayed out of the outer membrane.

Modifying and Improving the Existed Biobrick

To be mentioned, we modified this commonly used transmembrane protein which has been submitted by Warsaw iGEM team in 2008, BBa_K103006 . The most contributed modification to this biobrick is that we replaced the long GS linker behide the transmembrane protein but we chose a six nucleotides restriction site, NcoI, to make it express remarkably on the outer membrane. This change has two benefits. First, the appearance of NcoI allowed the linked protein, for example scFv in our project, to be easily changed and to be brought outside conveniently (like a cassette). Second, NcoI also can act as an useful linker in fusion protein (between transmembrane protein and the linked protein), this has been proved in our project. Moreover, we also selected the amino acids of OmpA from the forty-sixth till the one hundred fifty-ninth and the function of Lpp-OmpA to bring scFv onto the outer membrane E. coli is completely proved by our experiments.

An alternative version with BamHI site

If you'd like to directly use this part as a vector/backbone for cloning a fusion protein, you should be noticed that an extra NcoI site is present on Cm resistance gene. BBa_K1991004 replaced NcoI site behind LO with BamHI site and provides an alternative choice for cloning a LO fusion protein


The features of Lpp-OmpA expression system


The versatile applications: The diversity of introduced protein connecting to OmpA can provide the multiple functions outside the surface of the E. coli. For instance, in our project, we can change every kind of scFv we desire.

The stabilization of expression: Due to the anchoring capabilities of lipoprotein and outer membrane protein, the Lpp-OmpA expression system can display passenger proteins more efficiently and also performs the strong adaptability to the various passenger protens.

Experiment

After receiving the DNA sequences from the gene synthesis company, we recombined each Lpp-OmpA-N gene to pSB1C3 backbones and conducted a PCR experiment to check the size of each of the Lpp-OmpA-N. The DNA sequence length of the Lpp-OmpA-N is around 400~500 bp. In this PCR experiment, the Lpp-OmpA-N products size should be near at 650~750 bp. Fig.2 showed the correct size of the Lpp-OmpA-N, and proved that we successful ligated the Lpp-OmpA-N sequence onto an ideal backbone.

Fig.2
A:Lpp-OmpA-NcoI The DNA sequence length of the Lpp-OmpA-N is around 400~550 bp. In this PCR experiment, the Lpp-OmpA-N products size should be close to 650~800 bp.
Fig.3 Plate of Lpp-OmpA-NcoI

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 388
  • 1000
    COMPATIBLE WITH RFC[1000]