Difference between revisions of "Part:BBa K4417013"

 
Line 3: Line 3:
 
<partinfo>BBa_K4417013 short</partinfo>
 
<partinfo>BBa_K4417013 short</partinfo>
  
−
aa
+
<h1>Description</h1>
 +
 
 +
The coding sequence of ureEFG (<partinfo>BBa_K4417010</partinfo>) was cloned into pCT5c (<partinfo>BBa_K4417000</partinfo>) and could be induced by cumate promoter (<partinfo>BBa_K4417007</partinfo>). Besides, this composite contains a strong RBS (<partinfo>BBa_K4417008</partinfo>) and an rrnB T1 terminator (<partinfo>BBa_K4417011</partinfo>). This composite part is the ureEFG gene from ''Sporosarcina pasteurii''.
 +
 
 +
ureEFG are urease (accessory) proteins, responsible for the activation of the ureABC proenzyme. ureEFG is responsible for the transport and assembly of the nickel II center.
 +
 
 +
[[File:Zjy38.png|600px|thumb|center|'''Figure 1:''' Composite part: CuO-RBS-ureEFG-rrnB T1 Terminator TU2.]]
 +
 
 +
<h1>Usage and Biology</h1>
 +
 
 +
* This composite part can be used to produce urease accessory proteins.
 +
* The part was synthesized by IDT as a gBlock.
 +
 
 +
<h1>Cloning Strategy</h1>
 +
 
 +
This part was flanked by SapI Type IIS prefix and suffix in order to facilitate sharing of the constructs among the scientific community. In addition, BamHI and SacI sites were used to clone this transcriptional unit into pCT5c plasmid using restriction enzyme digest.
 +
 
 +
[[File:Zjy39.png|600px|thumb|center|'''Figure 2:''' Construct: CuO-RBS-ureABC-rrnB T1 Terminator TU2.]]
 +
 
 +
Construct TU2 was ligated with ureEFG (<partinfo>BBa_K4417013</partinfo>) and pCT5c (<partinfo>BBa_K4417000</partinfo>). In Figure 2, the cloned plasmid was checked by diagnostic digest. ureEFG has a smaller size than  ureABC, indicated by the bottom band size deviation. Correct band size was observed with 6880bp and 2264bp.
 +
 
 +
[[File:Zjy30.png|600px|thumb|center|'''Figure 3:''' Diagnostic digest of construct TU2. 1: DNA ladder, 2: pCT5c cut with SapI, 3: Construct TU2 cut with SapI (6880bp, 2264bp), 4: Construct TU1 cut with SapI (6880bp, 2757bp).]]
 +
 
 +
Construct TU2 was further verified from Sanger sequencing.
 +
 
 +
 
  
−
<!-- Add more about the biology of this part here
 
−
===Usage and Biology===
 
  
 
<!-- -->
 
<!-- -->

Revision as of 11:35, 12 October 2022


CuO-RBS-ureEFG-rrnB T1 Terminator TU2

Description

The coding sequence of ureEFG (BBa_K4417010) was cloned into pCT5c (BBa_K4417000) and could be induced by cumate promoter (BBa_K4417007). Besides, this composite contains a strong RBS (BBa_K4417008) and an rrnB T1 terminator (BBa_K4417011). This composite part is the ureEFG gene from Sporosarcina pasteurii.

ureEFG are urease (accessory) proteins, responsible for the activation of the ureABC proenzyme. ureEFG is responsible for the transport and assembly of the nickel II center.

Figure 1: Composite part: CuO-RBS-ureEFG-rrnB T1 Terminator TU2.

Usage and Biology

  • This composite part can be used to produce urease accessory proteins.
  • The part was synthesized by IDT as a gBlock.

Cloning Strategy

This part was flanked by SapI Type IIS prefix and suffix in order to facilitate sharing of the constructs among the scientific community. In addition, BamHI and SacI sites were used to clone this transcriptional unit into pCT5c plasmid using restriction enzyme digest.

Figure 2: Construct: CuO-RBS-ureABC-rrnB T1 Terminator TU2.

Construct TU2 was ligated with ureEFG (BBa_K4417013) and pCT5c (BBa_K4417000). In Figure 2, the cloned plasmid was checked by diagnostic digest. ureEFG has a smaller size than ureABC, indicated by the bottom band size deviation. Correct band size was observed with 6880bp and 2264bp.

Figure 3: Diagnostic digest of construct TU2. 1: DNA ladder, 2: pCT5c cut with SapI, 3: Construct TU2 cut with SapI (6880bp, 2264bp), 4: Construct TU1 cut with SapI (6880bp, 2757bp).

Construct TU2 was further verified from Sanger sequencing.



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 1085
    Illegal XhoI site found at 1839
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]