True, prokaryotic DNA is not naked DNA. It does have a lot of proteins associated with it - just not the same level of compaction provided by associated structural organizers we see in eukaryotes. H-NS, NAPs, HUs, FIS and many more are great examples of prokaryotic DNA organizers. I should have been more careful with my choice of words (at the cost of over-simplification) in this video. Thanks for pointing it out!

Yes, it is part of my future plan :)

araC gene i.e. CDS (not drawn for most of the video but see 9:42) is downstream of the operator. The O2 does not interrupt the CDS; the operator is between araC promoter and the araC CDS.

@@theCrux oh! That makes it clear, thank you so much!

Yes, fusions even with a linker does not guarantee that partner proteins will work - it depends from protein to protein. In case the partner protein(s) is misfolded (regardless of fusion) then yes, it just hangs there. Chemokines for instance are not amenable to N-terminal engineering (even a single amino acid extension can be bad). Mini-motifs are generally found in C-terminal ends. C-terminus also gets a lot of PTM because of solubility and accessibility. I am not sure if I would say it does not contain "functional domains". The C-terminal is usually the exposed end (disordered and solvent accessible) so more often than not, it tolerates linkers well (but not always). Unless you have a specific reason to keep the partner proteins "stuck together" (say for single-molecule imaging, protein tracking, etc.) you may consider adding a 2A-peptide before or after the linker. I may have alluded to 2As as bi-cistronic systems in the past video(s).

@@theCrux Oh you are right I forget about things like C-terminal phosphorylation of RNApol II which they told us at molecular bio course and underestimated importance of the protein ends. Apparently I have a lot to refresh! Anyway thank you for the answer 😊.

Ah, yes, nice catch! You are correct, it will import into the nucleus.

Restriction digest would be the first thing to try before going for Sanger Seq. Colony PCR works too, but it is prone to false positives given high sensitivity + it needs primers/PCR reaction; so it can get relatively expensive than the other two options.

Do you mean Transformation (not transfection)? In that case, yes. You transform bacteria with the TOPO reaction, get colonies, get plasmids from a bunch of them and then screen for orientation. There are many ways to do so: Restriction digest, Sanger, PCR etc. This video on plasmids goes into screening methods: ru-vid.com/video/%D0%B2%D0%B8%D0%B4%D0%B5%D0%BE-2pAM-FTFI2c.html

@@theCrux Thank you! I saw the video, another incredible one! So, for screening for right direction, Colony PCR follow by Sanger sequencing are a good approach?

Yes, that seems reasonable (although most people would just stop after their colony PCR shows appropriate results; and Sanger only if they have a CDS and they care about ORFs/mutations etc.). More commonly, you would extract plasmid from a bunch of colonies (mini-prep) and then directly Sanger on the plasmid and/or do restriction digest (with this approach, colony PCR is not required). Colony PCR (followed by Sanger, if necessary) is a quicker alternative if you are screening for "many" colonies and therefore don't want to spend time/resources on mini-preps.

BsaI is a Type IIS restriction enzyme. It is just how all Type IIS enzymes work (binding and cutting locations are not the same). The upper and lower strand getting cut at different lengths is also a property of the enzymes. Some enzymes leave a longer overhang, others a shorter one; some enzymes don't even leave an overhang. You will find more details in the video on restriction enzymes. Link: ru-vid.com/video/%D0%B2%D0%B8%D0%B4%D0%B5%D0%BE-001BkPSM074.html

When talking about ORFs, Contiguous = Continuous or Uninterrupted Non-Contiguous = Discontinuous or Interrupted For translation, ribosomes expect mature mRNA and therefore the ORF to be uninterrupted - which is true for bacteria at levels since they don't have introns; their ORF in mRNA = ORF in DNA. Eukaryotes (for intron-containing genes) have interrupted ORFs at DNA level and at the immature RNA level. Eventually, post-splicing, interruptions (introns) are removed to result in a continuous or contiguous ORF, ready for ribosomes.

@@theCrux Got it. Thanks for explaining.

Glad to hear that the content is useful :) And yes, RNAi will be part of this "Genetic Engineering" series. I hope to have that video up by the end of this year.

Thanks for the suggestion. I do expect to cover Genetics and chromosome biology at some point. Currently caught up in videos on Genetic Engineering :)

@@theCrux thank you so much. will be looking forward to it!

Yep, videos on CRISPR are in the works - to be published soon :)

​Do you plan to describe how is the whole editing system assembled for eukaryotic cells including vectors?

Yes, I do plan to talk about vectors and editing system at a conceptual level... and maybe a short "How to design CRISPR vectors" video which I am still debating.

@@theCrux oh yes, this would be a great topic to tackle!

@@theCrux Thanks it would be great! Looking forward to see it.

The way you draw inner and outer does not matter - there is no such thing as inner and outer as it means nothing. H and L carry meaning because it represents the base information. As long as you follow H and L, you are good! You can flip H and L to inner and outer (and vice verse) - just make sure to keep the direction of promoters consistent.