RNA

Part:BBa_K3559022

Designed by: Scott Stacey, Maria Torra I Benach   Group: iGEM20_Imperial_College   (2020-10-21)


BioBrick_BASIC_gRNA_CDC19_3

A gRNA targeting the yeast CDC19 promoter.

Biology

CDC19 is the Saccharomyces cerevisiae gene encoding the enzyme Pyruvate Kinase (E.C.2.7.1.40). Pyruvate Kinase is a central metabolism enzyme which catalyses the reaction that converts phosphoenolpyruvate to pyruvate. Pyruvate is then fed into the TCA cycle (aerobic) or glucose fermentation (anaerobic).

This particular gRNA targets 113bp upstream from the transcription start site of CDC19, as it is intended to target close to the CDC19 promoter for maximal transcriptional knockdown.

An additionally interesting piece of biology is the ribozymes contained within this sequence. These are RNA motifs that form secondary structures with catalytic activity. The specific ribozymes in this part the Hammerhead and HDV ribozymes, can carry out cleavage reactions at a specific location within their own RNA molecule. This makes these sequences useful for insulating parts from their transcriptional context to ensure they function as intended.

Usage

This part was aimed to be used in Imperial College's 2020 iGEM Tryptophan optimisation project. We reasoned that knocking down central metabolism in this way would direct flux towards the shikimate pathway and thereby increase accumulation of L-tryptophan. We believe this part would be of interest to any teams looking to engineer central metabolism in yeast.

Characterisation

As a result of restricted lab access due to the Covid pandemic, we were unable to test this part in the lab. We therefore can make no comment on whether this part functions as intended.

Sequence and Features

While we expect part will function as intended using BioBrick assembly as a single part, there are a number of additional features within the sequence. Firstly, the part is immediately flanked by the BASIC integrated prefix (iP) and the BASIC integrated suffix (iS), thus allowing teams to make use of BASIC assembly as opposed to BioBrick assembly should they wish. Secondly, the sgRNA itself is surrounded by a hammerhead ribozyme and an HDV ribozyme. This means that post transcriptionally the sgRNA will be cleaved from the remainder of the transcript. This means multiple of these type parts could be assembled together to produce gRNA arrays, where several gRNAs are transcribed and then released from the original transcript via the ribozyme activity. We therefore believe these parts are powerful tools for allowing easy multiplexed transcriptional regulation.


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 163
    Illegal NgoMIV site found at 192
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 8
    Illegal BsaI.rc site found at 236


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