Difference between revisions of "Part:BBa J04500"
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===Contribution made by SHSID 2020===
 
===Contribution made by SHSID 2020===
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The backbone for BBa_K3521005 was derived from pET-28a vector. The recombinant plasmid was transformed into BL21 (DE3) competent cells and induced with IPTG. After induction, a specific protein band that is consistent with the theoretical molecular weight of FnCpf1 was detected in SDS-PAGE (Figure 1). The recombinant FnCpf1 was successfully purified by Ni-affinity chromatography. 
 
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[[File:T--SHSID-BBa_K3521005 Fig 1.jpg]]
 
  
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Figure 1. SDS-PAGE analysis of FnCpf1 production in BL21 (DE3) cells. After induction, cells were collected by centrifugation and broken by sonication. The resulting supernatants were loaded to the PAGE gel with different amounts. Lane2 1-4: the samples from the cultures induced with 1 mM IPTG; Lane2 5-8: the samples from the cultures induced with 0.5 IPTG; lane 9: protein molecular weight standards.
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===Recombinant FnCpf1 production, purification, and SDS-PAGE analysis===
 +
 
 +
The backbone for BBa_K3521005 was derived from pET-28a vector. The recombinant plasmid was transformed into BL21 (DE3) competent cells and induced with IPTG. After induction, a specific protein band that is consistent with the theoretical molecular weight of FnCpf1 was detected in SDS-PAGE (Figure 2). The recombinant FnCpf1 was successfully purified by Ni-affinity chromatography. 
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[[File:T--SHSID-BBa_K3521005 Fig 0.jpg|600px|thumb|left|Figure 1. pET28a-Cas12a(FnCpf1)]]
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[[File:T--SHSID-BBa_K3521005 Fig 1.jpg|600px|thumb|left|Figure 2. SDS-PAGE analysis of FnCpf1 production in BL21 (DE3) cells. After induction, cells were collected by centrifugation and broken by sonication. The resulting supernatants were loaded to the PAGE gel with different amounts. Lane2 1-4: the samples from the cultures induced with 1 mM IPTG; Lane2 5-8: the samples from the cultures induced with 0.5 IPTG; lane 9: protein molecular weight standards.]]

Revision as of 08:25, 26 October 2020


IPTG inducible promoter with RBS

R0010.B0034


Usage and Biology

R0010 and B0034 will be digested and ligated together in the manner described on the "Registry of Standard Biological Parts" website.

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]


Contribution made by SHSID 2020

Recombinant FnCpf1 production, purification, and SDS-PAGE analysis

The backbone for BBa_K3521005 was derived from pET-28a vector. The recombinant plasmid was transformed into BL21 (DE3) competent cells and induced with IPTG. After induction, a specific protein band that is consistent with the theoretical molecular weight of FnCpf1 was detected in SDS-PAGE (Figure 2). The recombinant FnCpf1 was successfully purified by Ni-affinity chromatography.

Figure 1. pET28a-Cas12a(FnCpf1)
Figure 2. SDS-PAGE analysis of FnCpf1 production in BL21 (DE3) cells. After induction, cells were collected by centrifugation and broken by sonication. The resulting supernatants were loaded to the PAGE gel with different amounts. Lane2 1-4: the samples from the cultures induced with 1 mM IPTG; Lane2 5-8: the samples from the cultures induced with 0.5 IPTG; lane 9: protein molecular weight standards.