Difference between revisions of "Part:BBa K4408010"

 
Line 3: Line 3:
 
<partinfo>BBa_K4408010 short</partinfo>
 
<partinfo>BBa_K4408010 short</partinfo>
  
−
1
+
This part is responsible for expressing adhE2 protein, SpyCatcher-thl fusion protein and SpyTag-hbd fusion protein. The expression of these proteins are simultaneously controlled by a Pthl promoter with a ribosome binding site (RBS) and a Cpa fdx terminator. It consists of BBa_K3443002(Pthl), BBa_K103015 (RBS1), BBa_K1462060(adhE2), BBa_K4408005(SpyCatcher-Linker 1-thl), BBa_K4408006 (SpyTag-Linker 1-hbd) and BBa_K3585002(Cpa fdx terminator). In our program, we use this device to construct and optimize the synthesis pathways of butanol and butyrate in Clostridium tyrobutyricum. By introducing adhE2，butyryl-CoA is converted to butyl aldehyde and further transformed to butanol. SpyCatcher-thl fusion protein and SpyTag-hbd fusion protein can bound together via the SpyCatcher/SpyTag system. This improves the efficiency of the reactions from acetyl-CoA to 3-hydroxybutytyl-CoA catalyzed by thl and hbd enzymes, which is a key step in the production of butyrate. Pthl starts the transcription and Cpa fdx terminator ends the transcription.
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
Line 11: Line 11:
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K4408010 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4408010 SequenceAndFeatures</partinfo>
−
 
+
===Results===
 +
pMTL-Pthl-adhE2 recombinant plasmid (refer to: BBa_K4408008 ) was digested with XbaI enzyme to obtain the linear vector. Using C. tyrobutyricum genome as template, PCR amplification was performed by introducing SpyCatcher and SpyTag sequences through primers, resulting in fragment Spythl (1182bp) (i.e., SpyCatcher-thl) and fragment Spyhbd (849bp) (i.e., SpyTag-hbd). The fragment Spythl was ligated with the linear vector pMTL-Pthl-adhE2 by Gibson assembly method. pMTL-Pthl-adhE2-Spythl was transformed into E. coli JM109 strain. Colony PCR and DNA electrophoresis was performed to confirm the positive colonies. These colonies were transferred and expanded. Plasmids extracted from the colonies were confirmed to be pMTL-Pthl-adhE2-Spythl by gene sequencing.
 +
The linear vector pMTL-Pthl-adhE2-Spythl was obtained by reverse PCR, and ligated with the fragment Spyhbd by Gibson assembly method. The correct pMTL-Pthl-adhE2-SpyCatcher-thl-SpyTag-hbd recombinant plasmid was obtained and verified by the same method as for pMTL-Pthl-adhE2-Spythl. The recombinant plasmid was also verified by enzymatic cleavage using Hind III enzyme.
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Revision as of 11:28, 1 October 2022


Pthl-adhE2-Spythl-Spyhbd, Expression of adhE2, thl and hbd, modified by SpyCatcher/SpyTag oligopept

This part is responsible for expressing adhE2 protein, SpyCatcher-thl fusion protein and SpyTag-hbd fusion protein. The expression of these proteins are simultaneously controlled by a Pthl promoter with a ribosome binding site (RBS) and a Cpa fdx terminator. It consists of BBa_K3443002(Pthl), BBa_K103015 (RBS1), BBa_K1462060(adhE2), BBa_K4408005(SpyCatcher-Linker 1-thl), BBa_K4408006 (SpyTag-Linker 1-hbd) and BBa_K3585002(Cpa fdx terminator). In our program, we use this device to construct and optimize the synthesis pathways of butanol and butyrate in Clostridium tyrobutyricum. By introducing adhE2，butyryl-CoA is converted to butyl aldehyde and further transformed to butanol. SpyCatcher-thl fusion protein and SpyTag-hbd fusion protein can bound together via the SpyCatcher/SpyTag system. This improves the efficiency of the reactions from acetyl-CoA to 3-hydroxybutytyl-CoA catalyzed by thl and hbd enzymes, which is a key step in the production of butyrate. Pthl starts the transcription and Cpa fdx terminator ends the transcription.

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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 4292
    Illegal SapI.rc site found at 4116

Results

pMTL-Pthl-adhE2 recombinant plasmid (refer to: BBa_K4408008 ) was digested with XbaI enzyme to obtain the linear vector. Using C. tyrobutyricum genome as template, PCR amplification was performed by introducing SpyCatcher and SpyTag sequences through primers, resulting in fragment Spythl (1182bp) (i.e., SpyCatcher-thl) and fragment Spyhbd (849bp) (i.e., SpyTag-hbd). The fragment Spythl was ligated with the linear vector pMTL-Pthl-adhE2 by Gibson assembly method. pMTL-Pthl-adhE2-Spythl was transformed into E. coli JM109 strain. Colony PCR and DNA electrophoresis was performed to confirm the positive colonies. These colonies were transferred and expanded. Plasmids extracted from the colonies were confirmed to be pMTL-Pthl-adhE2-Spythl by gene sequencing. The linear vector pMTL-Pthl-adhE2-Spythl was obtained by reverse PCR, and ligated with the fragment Spyhbd by Gibson assembly method. The correct pMTL-Pthl-adhE2-SpyCatcher-thl-SpyTag-hbd recombinant plasmid was obtained and verified by the same method as for pMTL-Pthl-adhE2-Spythl. The recombinant plasmid was also verified by enzymatic cleavage using Hind III enzyme.