(6 points possible)(Challenging)
We are now ready to choose the biological parts to implement an abstract GRN! You will use one of the GRNs that you found suitable in the previous exercise as the basis for this exercise.
To figure out the parts you're going to use, you look into your parts library and find the following biological components:
- CMV (cytomegalovirus) promoter: A constitutive promoter
- TetO: operator sequence which can be appended to a promoter to make a TetR-repressible promoter
- TetR: represses promoters appended with the TetO operator, unless bound by the small molecule dox (doxycycline)
- LacO1: operator which can be appended to a promoter to make a LacI-repressible promoter
- LacI: represses promoters appended with the LacO1 operator, unless bound by the small molecule IPTG
- shFF4: short hairpin RNA (a class of miRNA) that can be coded in the intron of a gene. The intron is spliced out (in the nucleus), the shRNA is folded into a hairpin, and is processed by RNAi machinery. In the cytosol it promotes the degradation of mRNA molecules bearing the FF4 target site (usually the 3' UTR is used)
- FF4bind: complementary sequence to FF4 shRNA that can be inserted into the 3' UTR of a transcript for RNAi
- shFF5 and target binding sites: same RNAi mechanism as FF4 but the sequence is different, making FF4 and FF5 mostly orthogonal
- poly A signal sequence: this sequences signals the addition of a poly A tail to a transcript and is relevant for the translation and stability of transcripts in eukaryotic cells. We have already added this signal sequence to the constructs, it is marked as "PAS"
- IRES: internal ribosome entry site which can be placed between two coding sequences following a single promoter. This allows the translation of two proteins from a single promoter
- 2A linker: "self-cleaving" small peptide. When placed between two proteins, the proteins are translated as a single peptide. The 2A linker self-cleaves, producing to protein chains, again resulting in expression of two proteins from a single promoter
- LacZ: produces the β-galactosidase enzyme for use in an enzymatic assay with X-gal
- GFP: green fluorescent protein
- mKate: red fluorescent protein
- hBax: a killer protein
With a subset of these parts, construct a two state circuit (mKate on, GFP off; mKate off, GFP on) by dragging the parts onto the DNA circuit diagram below. The inputs to your circuit should be dox and shFF4.
- you might not need all the parts below and not all the boxes need to be filled for a correct answer
- place any promoters in the box directly beneath the promoter arrow and any operator sequences in the box downstream of the promoter arrow
- put intronic shRNA-s to the 3' (right) of the gene they are introns of
That being said, you're 4th point is a subtle, but very important one. Whenever using miRNAs to compute logic, one must think about the mechanism behind it. Due to the spatiotemporal separation of RNA processing and miRNA repression, it turns out miRNA cannot be used to repress another miRNA!