Part:BBa_K4770021

Cp_GS_Chloroplast_Glutamate_Synthetase_Level_1

Description and Biology

Level 1 vectors are single transcriptional units. This Level 1 contains a constitutive promoter PPSAD, a transit peptide for the chloroplast, a coding sequence for the Glutamate Synthetase in Chlamydomonas reinhardtii linked through F2A (a self-cleaving peptide) to a NanoLuc reporter. With this vector we aim to enhance Cp-GS expression, making the last step of the nitrates assimilation pathway (from nitrites to ammonium) constitutive by using the PPSAD promoter and being able to measure this expression through the NanoLuc signal.

How can this part be used

This Level 1 is aimed to later be assembled in a level M in position 5 through MoClos assembly. Should you use this part and need advice on how to use it, please do not hesitate to contact us at algagenix@gmail.com

Source of this part

- Original PPSAD sequence: BBa_K4770007-

- Original Cp sequence: BBa_K4770010

- Original GS sequence: BBa_K4770004

- Original F2A sequence: BBa_K4770012

- Original NanoLuc sequence: BBa_K4770011

- Original TPSAD sequence: BBa_K4770008

Design considerations

We performed Chlamydomonas reinhardtii's domestication for GS sequence. This process includes a reduction of the GC% content to make the sequence suitable for commercial synthesis while maintaining it high enough to fit Chlamydomonas reinhardtii's codon usage. This was done using our optimizing software (see AlgaGenix's wiki for additional information). Moreover, recognition sites for BbsI and BsaI were eliminated, changing said codons with synonymous ones. Besides, we also added an intron to perform what is known as intron-mediated enhancement (IME), which studies show aids with stable high-level expression of a foreign gene (Lumbreras et al., 1998) AlgaGenix’s Level 1s are designed to have the same structure and not having to build different pieces with different positions for each gene.

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 2473
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 2473
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 2473
  Illegal BglII site found at 2811
  Illegal BamHI site found at 2117
  Illegal XhoI site found at 2329
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 2473
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 2473
  Illegal NgoMIV site found at 1675
  Illegal NgoMIV site found at 2627
  
• 1000
  COMPATIBLE WITH RFC[1000]