Difference between revisions of "Part:BBa K1927001"
 
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===Usage and biology===
 
This part sequence is collected from the strain K71-77 Esterichia Coli and the gene is called
 
This part sequence is collected from the strain K71-77 Esterichia Coli and the gene is called
 
blaCMY-6 class C plasmid – mediated AmpC.
 
blaCMY-6 class C plasmid – mediated AmpC.
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been named AmpC in 1940. (1)
 
been named AmpC in 1940. (1)
  
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===Characterization of BB_K1927001===
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<p class="boxnotes leftext"><b>Generation of biobrick BBa_K1927001</b><br>
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This biobricks sequence is collected from a clinical isolate and it’s called blaCMY – 6 plasmid – mediated amp.
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This represents a class C enzyme from the broad family of β – lactamases.
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</p>
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  <p class="boxnotes leftext">The gene was designed in the same way as BBa_K1927000
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(see generation of BBa_K1927000) and is designed with specific flanking regions and without promoter for safety reasons.</p>
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[[File:T--UiOslo_Norway--plate001.png|700px|thumb|center|Figure 1:<i> Result from Gibson Assembly. Transformed G.A product into TOP10 chemically competent cells. Colonies grown on chloramphenicol plates.</i>]]
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  <p class="boxnotes leftext imgcap">To confirm that the insert is present we had the plasmid digested with XbaI and PstI along with BBa_K1927000 as a positive control.</p>
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[[File:T--UiOslo_Norway--gelpic.png|700px|thumb|center|Figure 4:<br><i>Lane 1: 1kb ladder gene ruler<br>
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Lane 2: BBa_K1927000 cut w/XbaI and SpeI<br>
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Lane 3:BBa_K1927001 cut w/XbaI and SpeI</i>]]
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  <p>The cut insert shows band at 1000bp which confirms the generation of the desired construct.
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</p>
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References:
 
References:
  
 
(1) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2620637/
 
(1) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2620637/

Latest revision as of 20:47, 30 October 2016

Usage and biology

This part sequence is collected from the strain K71-77 Esterichia Coli and the gene is called blaCMY-6 class C plasmid – mediated AmpC. These AmpC - lactamases mediate resistance to different antibiotics such as cephalothin, cefazolin, cefoxitin, most penicillins and also - lactamase inhibitor - lactam combinations.(1) They have also the ability to become resistance to several broad spectrum cephalosporins upon therapy because of overexpression of the gene and high mutation rate. The gene can both exist chromosomally and on plasmids. Techniques to identify AmpC - lactamases exist but are not yet optimized for the clinical laboratory. Even though the prevalence of these bacteria is lower compared to other ESBL producing bacteria in the world one should not underestimate its mechanism and it may be both harder to detect and broader in spectrum. This type of gene was also the first to be reported to neutralize penicillin, although it had not been named AmpC in 1940. (1)

Characterization of BB_K1927001

Generation of biobrick BBa_K1927001
This biobricks sequence is collected from a clinical isolate and it’s called blaCMY – 6 plasmid – mediated amp. This represents a class C enzyme from the broad family of β – lactamases.

The gene was designed in the same way as BBa_K1927000 (see generation of BBa_K1927000) and is designed with specific flanking regions and without promoter for safety reasons.

Figure 1: Result from Gibson Assembly. Transformed G.A product into TOP10 chemically competent cells. Colonies grown on chloramphenicol plates.

To confirm that the insert is present we had the plasmid digested with XbaI and PstI along with BBa_K1927000 as a positive control.


Figure 4:
Lane 1: 1kb ladder gene ruler
Lane 2: BBa_K1927000 cut w/XbaI and SpeI
Lane 3:BBa_K1927001 cut w/XbaI and SpeI

The cut insert shows band at 1000bp which confirms the generation of the desired construct.

References:

(1) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2620637/