Using CRISPR Gene Editing to Modify the LacZ gene in E.coli

In this investigation, CRISPR-Cas9 we be used to cut bacterial chromosomal DNA at a specific location within the lacZ gene. You will then take advantage of the cells’ ability to perform HDR to cause a desired change in the lacZ sequence. You will do this by providing the cells with large quantities of a donor template DNA, which includes an insert with a stop codon that will disrupt the gene function. In short, the chromosomal lacZ gene will be edited in E. coli by transforming them with plasmid-based expression system for donor template DNA and guide RNA.

[lynnwade]

This was so well explained. Very thankful for this. Makes wish all my professors explained topics so efficiently. I truly understand this well now!

[starmer]

Thanks man. I'm giving a presentation on this exact experiment to my entire bio dept (students and professors) on what I found doing this experiment and I was having a real hard time explaining HDR and homologous recombination. How you explained it gave me a "aha moment". Thanks!!!!

[refad]

Such an important and awesome session. Thanks a lot for the presentation.

[j_rzd]

Thank you so much sir for providing such a detailed presentation, you have saved my life! :)

[ilyesbgtf]

Thanks Professor, Its been so useful for me !

[Inferno]

This is a great video. I would like to do an experiment for lab genetics lab but i am unsure of what kind of experiment to run. I am not sure what could be changed or added to or what questions can be asked. Do you have any ideas or examples of experiments that can be done with this technology?

[felixmetzger]

I would think that A should become blue since there is no cut (because of the lack of sgRNA) and B should stay colorless or even die because the LacZ gene will be cut but not repaired since the lack of Arabinose. And since the sgRNA is missing for the cut C should remain colorless. If I got it right only D should successfully perform the insert of the stop codon which should end up as a colorless medium.
Is this correct?

[sugapriya]

Hi .. can we find what kind of modification has happened in CRISPR sequence ...that is how to compare the target with the edited sequence

[ilyesbgtf]

Hello Professor,

Is this right ?

A: Blue
B:No colony
C:Blue
D:White