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stop codon readthrough in E.coli

stop codon

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#1 Biogareth

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Posted 30 August 2011 - 09:16 AM

Dear all,

Recently I found one of my mutants contains stop codon before 100 aminoacid to the end. When I checked the expression by SDS-page, and found there are two equally dense bands (one supposed to be the partial protein and the other is the full protein).
The stop codon for my case is TAG. I know it is reasonable it can be bypassed with stopcodon.
Now I am wondering which amino acid will be translated to in case of stop codon readthrough? and Which strategy can be applied to make the stop codon efficiently to stop expression fully at that position?
Thanks in advance!

Any suggestion is welcome!

Biogareth

#2 pDNA

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Posted 30 August 2011 - 11:57 AM

what strain do you use?

...normally UAG should be a stop codon ...no way round!
maybe you have a frame shift or an alternative ATG near the RBS that could initiate translation into another frame?

do you cultivate at high temperatures (42°C) or do you use a special mutant strains (prfA?)?

Regards,
p

#3 Biogareth

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Posted 30 August 2011 - 11:23 PM

what strain do you use?

...normally UAG should be a stop codon ...no way round!
maybe you have a frame shift or an alternative ATG near the RBS that could initiate translation into another frame?

do you cultivate at high temperatures (42°C) or do you use a special mutant strains (prfA?)?

Regards,
p



Hi,

The strain I used is XL10Gold competent cell, and the cultivation temperature is 37 degree.
I always use the same plasmid for mutation analysis, and there is no frame-shift for sure.
Thanks!

B

#4 pDNA

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Posted 31 August 2011 - 11:23 AM

maybe your culture is a mixed population? ...did you saw any sequence overlays at this position or was the base calling totally clear?

Regards,
p

#5 Biogareth

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Posted 31 August 2011 - 11:30 PM

maybe your culture is a mixed population? ...did you saw any sequence overlays at this position or was the base calling totally clear?

Regards,
p



Yes, before we did expression, sequencing charomatography showed it is only one nucleaotide without any contamination.

#6 prodes

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Posted 01 September 2011 - 08:32 AM

Hi,

It could be Serine, Leucine, Glutamine or Tyrosine.

http://ecoliwiki.net...nse_suppressors

Cheers

#7 Biogareth

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Posted 01 September 2011 - 12:20 PM

Thanks

Hi,

It could be Serine, Leucine, Glutamine or Tyrosine.

http://ecoliwiki.net...nse_suppressors

Cheers



Thanks. It is useful.

#8 pDNA

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Posted 02 September 2011 - 06:08 AM

true!!! ...when you take a close look at the XL10Gold genotype (see here) you'll find that XL10Gold is glnV44, meaning that amber (UAG) stop codons are suppressed by insertion of glutamine, that particular genotype is required for some phage growth.

Problem solved Posted Image

Regards,
p

Edited by pDNA, 02 September 2011 - 06:09 AM.


#9 Biogareth

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Posted 02 September 2011 - 07:27 AM

true!!! ...when you take a close look at the XL10Gold genotype (see here) you'll find that XL10Gold is glnV44, meaning that amber (UAG) stop codons are suppressed by insertion of glutamine, that particular genotype is required for some phage growth.

Problem solved Posted Image

Regards,
p



It's great. I just found the explanation in few minute before.
Thanks!

Cheers!





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