Difference between revisions of "Part:BBa K2933259"
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===Usage and Biology===
 
===Usage and Biology===
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PST-1 is a type of subclass B1 metal beta-lactamases. The beta lactamases of subclass B1 family can hydrolyze almost all available beta lactam antibiotics (except aztreonam) clinically, including the broad-spectrum antibiotic carbapenems. Because of the extensive substrate profile of this enzyme, the clinical strains carrying it become a great threat to human life and health.<br>
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This composite part is made up with eight basic parts, T7 Ribosome binding sites,the His-Sumo tag, three cutting sites of Prescission Protease, our target protein PST-1 and T7 terminator. It encodes a protein which is PST-1 fused with His-Sumo tag. The fusion protein is about 38.2 kD. The fusion protein can be cut off at the cutting sites by Prescission Protease. It is convenient for us to purify our target protein.
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===Molecular cloning===
 
===Molecular cloning===
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First, we used the vector pET28B-Sumo to construct our expression plasmid. And then we converted the plasmid constructed to ''E. coli'' DH5α to expand the plasmid largely.<br>
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First, we used the vector pET-28bs to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.
 
<p style="text-align: center;">
 
<p style="text-align: center;">
 
   [[File:PST-1-PCR.png]]<br>
 
   [[File:PST-1-PCR.png]]<br>
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'''Affinity Chromatography:'''<br>
 
'''Affinity Chromatography:'''<br>
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We used the Ni-sepharose to purify the target protein. The Ni-sepharose can combine specifically with the His tag fused with target protein. <br>
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We used the His-Sumop sepharose to purify the target protein. The Ni-sepharose can combine specifically with the His-Sumo tag fused with target protein. <br>
 
* First, wash the column with water for 10 minutes. Change to Ni-binding buffer for another 10 minutes and balance the Ni column.<br>
 
* First, wash the column with water for 10 minutes. Change to Ni-binding buffer for another 10 minutes and balance the Ni column.<br>
 
* Second, add the protein solution to the column, let it flow naturally and bind to the column. <br>
 
* Second, add the protein solution to the column, let it flow naturally and bind to the column. <br>

Revision as of 13:50, 22 September 2019


RBS b+Linker h+His+Linker a+Sumo+Linker b+PST-1+T7 terminator

This part consists of RBS, protein coding sequence(His+Linker a+Sumo+Linker b+PST-1) and T7 terminator,and the biological module can be build into E.coli for protein expression. This part can be prefaced with promoters of different strengths and types to regulate expression function.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 298
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 298
    Illegal NheI site found at 75
    Illegal NheI site found at 1134
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 298
    Illegal BglII site found at 187
    Illegal BamHI site found at 386
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 298
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 298
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

This composite part is made up with eight basic parts, T7 Ribosome binding sites,the His-Sumo tag, three cutting sites of Prescission Protease, our target protein PST-1 and T7 terminator. It encodes a protein which is PST-1 fused with His-Sumo tag. The fusion protein is about 38.2 kD. The fusion protein can be cut off at the cutting sites by Prescission Protease. It is convenient for us to purify our target protein.

Molecular cloning

First, we used the vector pET-28bs to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.

PST-1-PCR.png
Figure 1. a: The PCR result of PST-1. b: The verification results by enzyme digestion.

After verification, it was determined that the construction is successful. We converted the plasmid to E. coli BL21(DE3) for expression and purification.

Expression and purification

Pre-expression:
The bacteria were cultured in 5mL LB liquid medium with ampicillin(100 μg/mL final concentration) in 37℃ overnight.
Massive expressing:
After taking samples, we transfered them into 1L LB medium and add antibiotic to 100 μg/mL final concentration. Grow them up in 37°C shaking incubator. Grow until an OD 600 nm of 0.8 to 1.2 (roughly 3-4 hours). Induce the culture to express protein by adding 1 mM IPTG (isopropylthiogalactoside, MW 238 g/mol). Put the liter flasks in 16°C shaking incubator for 16h.

Affinity Chromatography:
We used the His-Sumop sepharose to purify the target protein. The Ni-sepharose can combine specifically with the His-Sumo tag fused with target protein.

  • First, wash the column with water for 10 minutes. Change to Ni-binding buffer for another 10 minutes and balance the Ni column.
  • Second, add the protein solution to the column, let it flow naturally and bind to the column.
  • Third, add Ni-Washing buffer several times and let it flow. Take 5ul of wash solution and test with Coomassie Brilliant Blue. Stop washing when it doesn’t turn blue.
  • Forth, add Ni-Elution buffer several times. Check as above.
  • Fifth, collect the eluted proteins for further operation.

T--TJUSLS China--PST-1 Ni.png
Figure 2. The result of SDS-page.