Difference between revisions of "Part:BBa K2429063"

Latest revision as of 18:23, 27 October 2017

mKate FF4 Guide Family Member

Parts K2429063 through K2429068 and parts K2429083 through K2429093 are a family of guide sequences used by either a dCas13a variant or Ms2 to target the intron of the mKate FF4 reporters (BBa_K2429033 and BBa_K2429049). These guides lie downstream of a U6 promoter, which is a constitutive promoter typically used for the production of guide RNA.

Our team used multiple guide sequences to "tile" along the reporter’s intron to determine what part of the intron is best to target when trying to control its splicing efficiency. Based on the level of fluorescence associated with each guide, we can compare the results to see whether a specific targeted sequence is integral in the process of splicing, and whether the binding of the RNA binding protein to said sequence changes the splicing efficiency.

The guides shift by a few base pairs so we can make more precise conclusions about what intronic sequences are important for splicing.

Parts K-63 through K-68 lead to guide RNA sequences that serve as a guide for the dCas13a protein.

Parts K-83 through K-87 as well as K-93 lead to the production of antisense oligonucleotides (ASOs). Parts K-88 through K-92 consist of the same ASO sequences corresponding to the same order (i.e. K-83 sequence is used in K-88); however, they have an additional portion of nucleotides that will form a hairpin loop which Ms2 can bind. The addition of oligonucleotides without hairpins loops serves as a control to test whether it’s the guide sequence or the protein that is actually affecting splicing.

Part K-062 is a guide RNA targeting a regular mKate sequence. This guide serves as a control for all split mKate-targeting experiments.

Sequence and Features