What is L4440 plasmid?
In Caenorhabditis elegans, RNAi can be achieved by feeding worms bacteria carrying a plasmid expressing double-stranded RNA (dsRNA) targeting a gene of interest. The most commonly used plasmid vector for this purpose is L4440. These results indicate cross-regulation between different small RNA pathways in C.
What is Iptg in RNAi?
The IPTG will compete with the repressors on the lac o promoter and remove them and allow the gene for T7 RNA polymerase to be transcribed and then translated into the RNA polymerase protein. The T7 RNA polymerase then binds to the T7 promoters on the pL4440 plasmid and transcribes our C. elegans DNA into RNA!
How does RNAi feeding work?
In the feeding protocol, dsRNA is transcribed in E. coli and ingested by animals. The protocols provided in this unit are meant to help the complete novice at worm culture select and apply the appropriate RNAi delivery method.
Why do we need ampicillin to make RNAi work?
One thing we do know is that RNAi knockdown works better when you use freshly transformed feeding bacteria that can be manipulated to overexpress plasmid genes of interest, so that’s what we’ll do. You added ampicillin to the broth to ensure that the plasmid DNA would be maintained by the transformed cells.
How is RNAi done C elegans?
There are three ways to carry out RNAi in C. elegans: injection (Fire et al., 1998), soaking (Tabara et al., 1998), and feeding (Timmons and Fire, 1998). All three can produce efficient gene knockdowns.
Is the l4440 plasmid selectable for ampicillin resistance?
The plasmid also has a selectable marker that confers resistance to an antibiotic, in this case ampicillin. In 7.15, you will use the E. coli strain HT115 carrying various L4440 plasmids, each containing a different cloned gene sequence.
What kind of side chain does carbenicillin have?
Carbenicillin is a penicillin antibiotic having a 6beta-2-carboxy-2-phenylacetamido side-chain. It has a role as an antibacterial drug. It is a penicillin and a penicillin allergen.
How many RNAi feeding plates are needed for l4440?
You should have three RNAi feeding plates: a. One plate will be seeded with HT115 bacteria carrying the “empty” L4440 vector (no C. elegans gene is cloned in the vector). b. Another plate will be seeded with HT115 bacteria carrying a portion of the unc-22 gene cloned into the L4440 vector.
What kind of antibiotic is carbenicillin beta lactamase resistant?
Carbenicillin is a broad-spectrum, semi-synthetic penicillin antibiotic with bactericidal and beta-lactamase resistant activity. Carbenicillin acylates the penicillin-sensitive transpeptidase C-terminal domain by opening the lactam ring.
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