Difference between revisions of "Part:BBa K2933214"

 
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<partinfo>BBa_K2933214 parameters</partinfo>
 
<partinfo>BBa_K2933214 parameters</partinfo>
 
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==Usage and Biology===
 +
This composite part is made up with three basic parts, the His tag, T7 promoter, RBS and our target protein SHD. It encodes a protein which is SHD fused with His tag. The fusion protein is about 29.0 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of SHD .  It is convenient for us to purify our target protein.<br>
 +
===Molecular cloning===
 +
First, we used the vector pGEX-6p-1 to construct our expression plasmid. And then we converted the plasmid constructed to ''E. coli'' DH5α to expand the plasmid largely.<br>
 +
<p style="text-align: center;">
 +
  [[File:SHD-PCR.png|500px]]<br>
 +
'''Figure 1.'''  Left: The PCR result of SHD. Right: The verification results by enzyme digestion.<br>
 +
</p>
 +
After verification, it was determined that the construction is successful. We converted the plasmid to ''E. coli'' BL21(DE3) for expression and purification.<br>
 +
 +
 +
===Expression and purification===
 +
'''Pre-expression:'''<br>
 +
The bacteria were cultured in 5mL LB liquid medium with ampicillin(100 μg/mL final concentration) in 37℃ overnight.<br>
 +
'''Massive expressing:'''<br>
 +
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.<br>
 +
 +
'''Affinity Chromatography:'''<br>
 +
We used the GST Agarose to purify the target protein. The GST Agarose can combine specifically with the GST tag fused with target protein. <br>
 +
* First, wash the column with GST-binding buffer for 10 minutes to balance the GST column.<br>
 +
* Second, add the protein solution to the column, let it flow naturally and bind to the column.<br>
 +
* Third, add GST-Washing buffer several times and let it flow. Take 10μl of wash solution and test with Coomassie Brilliant Blue. Stop washing when it doesn’t turn blue.<br>
 +
* Forth, add 400μL Prescission Protease (1mg/mL) to the agarose. Digest for 16 hours in 4℃.
 +
* Fifth, add GST-Elution buffer several times. Check as above. Collect the eluted proteins for further operation.<br>
 +
<p style="text-align: center;">
 +
    [[File:T--TJUSLS China--SHD GST.jpg|400px]]<br>
 +
 +
'''Figure 2.'''  The result of SDS-page.<br>
 +
</p>

Revision as of 13:57, 21 September 2019


T7 promoter+RBS b+Linker h+His+Linker f+SHD+T7 terminator

This part consists of T7 promoter, RBS and protein coding sequence（His+Linker f+SHD），and the biological module can be built into E.coli for protein expression

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 47
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 169
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 47
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 47
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology=

This composite part is made up with three basic parts, the His tag, T7 promoter, RBS and our target protein SHD. It encodes a protein which is SHD fused with His tag. The fusion protein is about 29.0 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of SHD . It is convenient for us to purify our target protein.

Molecular cloning

First, we used the vector pGEX-6p-1 to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.

SHD-PCR.png
Figure 1. Left: The PCR result of SHD. Right: 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 GST Agarose to purify the target protein. The GST Agarose can combine specifically with the GST tag fused with target protein.

  • First, wash the column with GST-binding buffer for 10 minutes to balance the GST column.
  • Second, add the protein solution to the column, let it flow naturally and bind to the column.
  • Third, add GST-Washing buffer several times and let it flow. Take 10μl of wash solution and test with Coomassie Brilliant Blue. Stop washing when it doesn’t turn blue.
  • Forth, add 400μL Prescission Protease (1mg/mL) to the agarose. Digest for 16 hours in 4℃.
  • Fifth, add GST-Elution buffer several times. Check as above. Collect the eluted proteins for further operation.

T--TJUSLS China--SHD GST.jpg
Figure 2. The result of SDS-page.