Difference between revisions of "Part:BBa K2933018"
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<partinfo>BBa_K2933018 parameters</partinfo>
 
<partinfo>BBa_K2933018 parameters</partinfo>
 
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===Usage and Biology===
 +
ARL-1 is a type of subclass A beta-lactamases, which is separated from Staphylococcus arlettae. The blaARL-1 was recently discovered and reported, that have a high mutation.<br>
 +
===Molecular cloning===
 +
First, we used the vector pET-28a(+) and the vector pET28a-SUMO 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:ARL-1-PCR.png|500px]]<br>
 +
'''Figure 1.'''  Left: The PCR result of ARL-1. 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 kanamycin(50 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>

Latest revision as of 07:46, 24 September 2019


subclass A metallo-beta-lactamase ARL-1, codon optimized in E. coli

This part encodes a protein called ARL-1, which is a beta-lactamase of subclass A.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 427
    Illegal PstI site found at 541
    Illegal PstI site found at 592
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 427
    Illegal PstI site found at 541
    Illegal PstI site found at 592
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 427
    Illegal PstI site found at 541
    Illegal PstI site found at 592
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 427
    Illegal PstI site found at 541
    Illegal PstI site found at 592
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

ARL-1 is a type of subclass A beta-lactamases, which is separated from Staphylococcus arlettae. The blaARL-1 was recently discovered and reported, that have a high mutation.

Molecular cloning

First, we used the vector pET-28a(+) and the vector pET28a-SUMO to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.

ARL-1-PCR.png
Figure 1. Left: The PCR result of ARL-1. 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 kanamycin(50 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.