Part:BBa_K3559017:Design
BioBrick_BASIC_gRNA_Aro8_1
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 163
Illegal NgoMIV site found at 192 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 8
Illegal BsaI.rc site found at 236
Design Notes
Beyond adding the BASIC and BioBrick prefixes and suffixes the primary design considerations were in designing the ribozymes and the gRNA sequences. These ribozymes were added to allow the generation of gRNA arrays where multiple gRNAs could be multiplexed as a single part. The sequence of the first 6bp of the Hammerhead ribozyme was modified to be the reverse complement of the first 6bp of the 20bp gRNA target sequence. This was automated using a basic python script (https://github.com/scottstacey/iGEM-2020/blob/master/iGEM_gRNA.py).
As the purpose of these gRNAs was for gene expression knockdown, gRNA sequences were designed to target the promoter or close to the transcription start site for a particular gene. A further design consideration for the gRNAs was to design gRNAs that did not target places too close together in the yeast genome so that multiplex gRNA knockdown of a single gene was possible. This was tried to be kept to a minimum of 15 bp apart.
Source
gRNA was designed using Benchling's gRNA design tool. HDV and hammerhead ribozyme sequences were provided by Will Shaw from Tom Ellis's lab at imperial.