Difference between revisions of "Part:BBa K2933013"

 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2933013 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2933013 SequenceAndFeatures</partinfo>
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<!-- Uncomment this to enable Functional Parameter display
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===Functional Parameters===
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<partinfo>BBa_K2933013 parameters</partinfo>
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<!-- -->
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===Usage and Biology===
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<!-- -->
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2933016 SequenceAndFeatures</partinfo>
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===Molecular cloning===
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===Exploration of expression condition===
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  [[File:SHD-28a.jpeg|160px|left|]] 
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'''Figure 2.'''
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The result of SDS-PAGE. <br>
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Lane1, uninduced SHD-28a(29.0kD). <br>
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Lane2, 37°C induced SHD-28a(29.0kD) <br>
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with 0.5mM IPTG.<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 kanamycin(50 μg/mL final concentration) in 37℃ overnight.<br>
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'''Massive expressing:'''<br>
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After taking samples, we transfer them into 1L LB medium and add antibiotic to 50 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 0.5 mM IPTG (isopropylthiogalactoside, MW 238 g/mol) or ~0.1 gram per 1.5 liter flask. Put the liter flasks in 16°C shaking incubator for 16h. Centrifuge your bacteria in 500 mL bottles in the 4°C rotor at 4,000 rpm for 20 mins. Do this in batches until all your culture is spun down saving the cell pastes each time.<br>           
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'''Purification of GST fusion proteins:'''<br>
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==References==
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Revision as of 08:06, 8 September 2019


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

This part encodes a protein called VIM-66, which is a metallo-beta-lactamase of subclass B1.

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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 738
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Molecular cloning

Exploration of expression condition

File:SHD-28a.jpeg






Figure 2. The result of SDS-PAGE.
Lane1, uninduced SHD-28a(29.0kD).
Lane2, 37°C induced SHD-28a(29.0kD)
with 0.5mM IPTG.



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 transfer them into 1L LB medium and add antibiotic to 50 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 0.5 mM IPTG (isopropylthiogalactoside, MW 238 g/mol) or ~0.1 gram per 1.5 liter flask. Put the liter flasks in 16°C shaking incubator for 16h. Centrifuge your bacteria in 500 mL bottles in the 4°C rotor at 4,000 rpm for 20 mins. Do this in batches until all your culture is spun down saving the cell pastes each time.

Purification of GST fusion proteins:



References