Difference between revisions of "Part:BBa K2933008"
 
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===Usage and Biology===
 
===Usage and Biology===
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To be uploaded
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blaPEDO-1, a new carbapenem-hydrolyzing beta-lactamases, this enzyme has not yet emerged in clinical settings but constitute potential carbap- enem resistance determinants in pathogenic bacterial species, as demonstrated by their ability to confer resistance to ampi- cillin and various cephalosporins, as well as reduced suscepti- bility to carbapenems, once expressed in E. coli.
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==References==
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===References===
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[1]Dereje Dadi Gudeta, Valeria Bortolaia,The Soil Microbiota Harbors a Diversity of Carbapenem-Hydrolyzing β-Lactamases of Potential Clinical Relevance[J]，Antimicrobial Agents and chemotherapy,January 2016<br>
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===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>
 
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;">
 
<p style="text-align: center;">
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   [[File:PEDO-1-PCR.png|500px]]<br>
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   [[File:PEDO-1-PCR.png|400px]][[File:PEDO-1-enzyme digestion.png|400px]]<br>
 
'''Figure 1.'''  Left: The PCR result of PEDO-1. Right: The verification results by enzyme digestion.<br>
 
'''Figure 1.'''  Left: The PCR result of PEDO-1. Right: The verification results by enzyme digestion.<br>
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</p>
 
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After verification, it was determined that the construction is successful. We converted the plasmid to ''E. coli'' BL21(DE3) for expression and purification.<br>
 
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===Expression and purification===
 
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'''Pre-expression:'''<br>
 
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The bacteria were cultured in 5mL LB liquid medium with ampicillin(100 μg/mL final concentration) in 37℃ overnight.<br>
 
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'''Massive expressing:'''<br>
 
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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>
 
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'''Affinity Chromatography:'''<br>
 
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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>
 
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* First, wash the column with GST-binding buffer for 10 minutes to balance the GST column.<br>
 
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* Second, add the protein solution to the column, let it flow naturally and bind to the column.<br>
 
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* 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>
 
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* Forth, add 400μL Prescission Protease (1mg/mL) to the agarose. Digest for 16 hours in 4℃.
 
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* Fifth, add GST-Elution buffer several times. Check as above. Collect the eluted proteins for further operation.<br>
 
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<p style="text-align: center;">
 
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    [[File:T--TJUSLS China--PEDO-1 GST.jpg|400px]]<br>
 
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'''Figure 2.'''  The result of SDS-page.<br>
 
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</p>
 
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'''Anion exchange column:'''<br>
 
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According to the predicted pI of the protein and the pH of the ion-exchange column buffer, firstly select the appropriate ion exchange column (anion exchange column or cation exchange column). The pH of buffer should deviate from the isoelectric point of the protein. Since the isoelectric point of our protein is 5.88 in theory, we choose buffer pH of 7.4 and use anion exchange column for purification.
 
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The protein is concentrated with a 10KD concentration tube, and then the exchange buffer is used to exchange the protein to the ion-exchange liquid A. Finally, it is concentrated to less than 5ml by centrifuging at 4℃ and 3400rpm for 10 minutes in a high-speed centrifuge to remove insoluble substances and bubbles.
 
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Balance the selected column with liquid A. Through the AKTApure protein purification system, the samples are loaded to the column at a flow rate of 0.5ml/min, and continue washing for 5min. Gradually increase the content of liquid B in the column, change the salt concentration and then change the interaction between the sample and the column, and collect the corresponding eluent according to the position of the peak. Use SDS-PAGE to check the result.<br>
 
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<p style="text-align: center;">
 
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  [[File:T--TJUSLS China--PEDO-1 Q.jpg|400px]]<br>
 
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'''Figure 1.'''  The result of SDS-page of superdex75 Q column.<br>
 
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</p>
 
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'''Gel filtration chromatography:'''<br>
 
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The collected protein samples are concentrated in a 10 KD concentrating tube at a speed of 3400 rpm and concentrated for a certain time until the sample volume is 500 μl. At the same time, the superdex 200 column is equilibrated with a buffer to balance 1.2 column volumes. The sample is then loaded and 1.5 cylinders are eluted isocratically with buffer. Determine the state of protein aggregation based on the peak position and collect protein samples based on the results of running the gel.<br>
 
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</p>
 
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<p style="text-align: center;">
 
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  [[File:T--TJUSLS China--PEDO-1 gel jiaotu+fengtu.png|600px]]<br>
 
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'''Figure 1.'''  (a) The result of gel filtration used the superdex75 column with the AKTA system, which shows that the target protein is monomeric. (b) The result of SDS-PAGE. And the target protein is about 32.2kD.<br>
 
 
</p>
 
</p>

Latest revision as of 12:07, 23 September 2019


subclass B1 metallo-beta-lactamase PEDO-1, codon optimized in E. coli

This part encodes a protein called PEDO-1, which is a metallo-beta-lactamase of subclass B1.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 537
    Illegal PstI site found at 823
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 537
    Illegal PstI site found at 823
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 537
    Illegal PstI site found at 823
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 537
    Illegal PstI site found at 823
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

blaPEDO-1, a new carbapenem-hydrolyzing beta-lactamases, this enzyme has not yet emerged in clinical settings but constitute potential carbap- enem resistance determinants in pathogenic bacterial species, as demonstrated by their ability to confer resistance to ampi- cillin and various cephalosporins, as well as reduced suscepti- bility to carbapenems, once expressed in E. coli.

References

[1]Dereje Dadi Gudeta, Valeria Bortolaia,The Soil Microbiota Harbors a Diversity of Carbapenem-Hydrolyzing β-Lactamases of Potential Clinical Relevance[J]，Antimicrobial Agents and chemotherapy,January 2016

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.

PEDO-1-PCR.pngPEDO-1-enzyme digestion.png
Figure 1. Left: The PCR result of PEDO-1. Right: The verification results by enzyme digestion.