Difference between revisions of "Part:BBa K2933013"

(→‎Usage and Biology)
Line 3: Line 3:
 
<partinfo>BBa_K2933013 short</partinfo>
 
<partinfo>BBa_K2933013 short</partinfo>
  
−
This part encodes a protein called VIM-66, which is a metallo-beta-lactamase of subclass B1.
+
This part encodes a protein called SHD, which is a metallo-beta-lactamase of subclass B1.
 +
 
  
−
<!-- Add more about the biology of this part here
 
 
===Usage and Biology===
 
===Usage and Biology===
 +
 +
Metallo-beta-lactamase SHD .
 +
  
 
<!-- -->
 
<!-- -->
Line 13: Line 16:
  
  
−
<!-- Uncomment this to enable Functional Parameter display
 
−
===Functional Parameters===
 
−
<partinfo>BBa_K2933013 parameters</partinfo>
 
−
<!-- -->
 
−
===Usage and Biology===
 
−
 
−
 
−
<!-- -->
 
−
<span class='h3bb'>Sequence and Features</span>
 
−
<partinfo>BBa_K2933013 SequenceAndFeatures</partinfo>
 
  
 
===Molecular cloning===
 
===Molecular cloning===
Line 43: Line 36:
 
The result of SDS-PAGE. <br>
 
The result of SDS-PAGE. <br>
 
Lane1, uninduced SHD-28a(29.0kD). <br>
 
Lane1, uninduced SHD-28a(29.0kD). <br>
−
Lane2, 37°C induced SHD-28a(29.0kD) <br>
+
Lane2, 37°C induced SHD-28a(29.0kD)with 0.5mM IPTG.<br>
−
with 0.5mM IPTG.<br>
+
  
  
Line 62: Line 54:
  
 
==References==
 
==References==
 +
  
  

Revision as of 08:11, 8 September 2019


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

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


Usage and Biology

Metallo-beta-lactamase SHD .


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]


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