I have to clone a gene below its native promoter but it is part of an operon, so I need to amplify the two genes separately and clone it in two steps.
I found a plasmid in my lab which already has the same gene cloned downstream of the native promoter but after analyzing the sequence, I found that there are two additional bases between the promoter and the START codon apart from the restriction site in between. I can perform PCR and amplify both sequences together by designing primers but the additional bases are going to increase the distance between the promoter and the gene. Would that create major problems during transcription?
Thank you.
In general, it is best to avoid adding extra bases between the promoter and the gene.
The promoter is a region of DNA that binds to RNA polymerase and initiates transcription. If the distance between the promoter and the gene is too great, RNA polymerase may not be able to bind to the promoter and transcribe the gene. It is best to keep the distance as short as possible.
No, not necessarily. An additional base or two between the promoter and the gene is not necessarily a problem, as long as the promoter is still able to bind to the gene and initiate its transcription. Depending on the type of gene, the presence of additional bases may even be beneficial, as they can provide additional binding sites for transcription factors that help regulate the gene's expression.
As long as your UTR does not have another start codon you should be fine.
So long as the two bases are well beyond the promoter recognition sequence then it should not be a problem at all. However if those bases are between the ribosome binding site and the start codon then it could affect translation (but probably no effect on transcription). So it depends where they are.
The impact of having two additional bases between the promoter and the gene on transcription depends on the specific promoter and gene in question. In general, promoters have specific sequences and spacing requirements for optimal function, and changes to the spacing or sequence can affect promoter activity. However, the effect may not necessarily be negative, and in some cases, it may even enhance gene expression.
If the two additional bases do not significantly alter the spacing or sequence requirements of the promoter, they may have minimal impact on gene expression. However, it's difficult to predict the effect of the additional bases without further experimentation or knowledge of the specific promoter and gene in question.
It's advisable to use the native promoter whenever possible to ensure proper regulation of the gene's expression. However, if using the plasmid with the additional bases is the only option, it may be worth testing the gene expression to determine the impact of the additional bases on promoter activity.
These video playlists might be helpful to you:
https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE
https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw
https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Materials Science
- Plating