Part:BBa_K5115053

ribozyme connected hox and hyp, without hoxU

Introduction

This composite part combines all the subunits, except hoxU, of the hydrogenase in our ribozyme-assisted polycistronic co-expression system：pRAP. Actually, compared with ribozyme connected hox and hyp, without hoxF, there is only one difference: one deleted hoxF and another deleted hoxU. The deleted subunits are all used to link with EP. EP ligation of different subunits leads to different effects of hydrogenase's entry into the carboxysome, which further affects the effect of our overall design. After comparison between experiments of these two designs, we make sure that the hoxF-linking-EP one is more satisfying. So, to learn more about the final adopted part, please click on ribozyme connected hox and hyp, without hoxF.

Characterization

Agarose gel electrophoresis

Figure 1. Agarose gel electrophoresis of PCR products amplified from one E. coli (DH5α) colony. M: DNA Marker. Lanes 1,3-8: Amplification of specific regions corresponding to hoxF, hoxY, hoxH, hoxW, hoxI, hypA, and hypB, demonstrating successful assembly and integrity of the ribozyme-connected hox and hyp, without hoxU (no band in lane 2) as designed. Primers for these PCR are listed on https://2024.igem.wiki/fudan/parts.

Sequence and Features

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 654
  Illegal BglII site found at 1472
  Illegal BglII site found at 1765
  Illegal BamHI site found at 2059
  Illegal XhoI site found at 99
  Illegal XhoI site found at 2266
  Illegal XhoI site found at 4505
  Illegal XhoI site found at 6334
  Illegal XhoI site found at 7496
  Illegal XhoI site found at 9335
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 533
  Illegal NgoMIV site found at 1071
  Illegal NgoMIV site found at 1281
  Illegal NgoMIV site found at 1593
  Illegal NgoMIV site found at 2359
  Illegal NgoMIV site found at 3039
  Illegal NgoMIV site found at 5646
  Illegal AgeI site found at 6821
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 4159
  Illegal BsaI.rc site found at 702
  Illegal BsaI.rc site found at 1206