I think the translation starts from ATG located in your "spacer", 7 nt after rbs. Check the cloning region of pEt15b plasmid to see the translation start.

Hi Barak,

I had a similar problem recently, but my linker was too long between the RBS and the ATG and had too many GCs, which seem to be inhibitory.

I've used this tool:

https://salislab.net/software/reverse

It really helped me out to increase the translation rate.

You can simply insert your sequence and it calculates the approximate translation rate. Try to change your spacer length and sequence to see the influence on the translation rate.

Concerning your spacer between the T7p and the lac operator, I usually use around 6-7 bp. That always worked fine for me.

Good luck!

Best,

Filip

Thank u for your answers,

AIRAT- the original peptide is called "my.gene", and i added the "ATG" and a few nucleotide before in order to express it. sorry that i didn't mention that.

Filip- i've try "salislab" as u said. unfortunately, it didn't come with any results... (see attach). although the website looks really cool!

I think i will try to add Gblock- in order to add a 30 b.p. spacer, hope it would solve my problem.

Hi Barak,

did you try the "Predict: RBS Translation rates" link on the left-hand side?

There you need to copy your DNA sequence beginning from right after the third G of the T7-Promotor sequence until the end of your gene of interest or until the end of the T7-Terminator sequence.

Then you need to choose an E.Coli strain or what ever organism you use to express and then it should be able to predict your Translation Rates.

I've tried it with the short sequence that you've provided and it showed a very low translation rate. ~95 for your ATG (see attachment). Another ATG at position 91 shows a higher translation rate than your ATG.. But considering that the rates sometimes go up to multiple tenthousands that is still miniscule. You might want to also use the "Design: RBS Sequence" tool to optimise your sequences.

The 30 bp spacer that you mentioned might also be a good way to start optimizing.

Best of luck,

Filip

Ah Sorry, it is "right before the three GGG of the T7 Promoter".

Hi Filip,

i'm not sure about my input data:

1.My pre-sequence is bigger than 20 b.p (45 b.p, includine the T7 promoter, lac. operator and the spacer- not include the RBS of course) , and the mark mentioned that i need to enter a sequence of 5-20 b.p- shall I enter the whole pre-seq.?

2. my protein seq length 2316 b.p. (start with the "ATG..." and ends up in temination seq. "...TAA"), but as I enter the whole seq. i can see that only a small part of it is submitted (limitation of the sali's lab program)- how can i be sure about the result if it only partially?

3. I mention that my "Target Translation Initiation Rate" is maximal (the goal), and i've used version 2.1 of the "free model energy".

after that being said...

3. I attach the results that i got from the program, as an excel format- to the right side is my gene, and on the left side is my gene with bigger spacer. if i did correct about the my input data- what do you think about those results? its seems that my initial translation rate is lower with the begger spacer, and yet, both are not in small numbers.

Thank you for your help, I'm really appreciate it!

Barak.