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There have been 5 items by donny (Search limited from 19-July 18)


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#160550 golden gate cloning

Posted by donny on 23 September 2013 - 01:58 AM in Molecular Biology

Not sure if you've read this

http://j5.jbei.org/j...l/pages/23.html

 

BsaI is a type II endonuclease and cuts somewhere downstream of its recognition site. It allows you to customise your overhanging end sequence. What you need to do is design primers such that the overhanging sequence is part of your gene sequence so that you don't introduce extra bases into your gene. For the adjacent gene, do the same with the complementary sequence on the complementary strand. Even though there is a base before the cleavage site, the base will be part of the fragment that you will be discarding. After BsaI digestion, you will get two fragments with complementary ends that will join at the junction of the two genes.

 

What do you mean by repetitive string of proteins? Fusion proteins? If so, you need to remove the TAA from the end of the gene.

 

Say, gene 1 ends with ACGGCCTAA and gene 2 starts with AGTTTC, you should design primers to get fragments like this (add extra bases to the ends of the primers for more efficient digestion).

 

Fragment for gene 1        Fragment for gene 2

....ACGGCCAGTTNGAGACC      GGTCTCNAGTTTC.....     

....TGCCGGTCAANCTCTGG      CCAGAGNTCAAAG.....

 

Since BsaI cuts like this (after the N's):

GGTCTCNXXXXX

CCAGAGNNNNNX

 

The resulting fragments will be:

 

Fragment for gene 1              Fragment for gene 2

....ACGGCC    AGTTNGAGACC      GGTCTCN    AGTTTC.....     

....TGCCGGTCAA    NCTCTGG      CCAGAGNTCAA    AG.....

 

See how the fragments at the far left and far right now have complementary sticky ends and fragments with the recognition site are discarded?

 

Hope that helps.

 

 

EDIT: Oops, it's been almost one month. Guess you've already figured out. Hope that helps anyway :)




#153643 Are two promoters needed for co-expressing 2 proteins from the same plasmid in S

Posted by donny on 12 April 2013 - 08:14 AM in Molecular Biology

Firstly, we place the promoter in front ofs the DNA sequence of the gene, not the other way round. Promoter sequence is where the RNA polymerase binds so as to transcribe the gene sequence into mRNA. The mRNA is then translated into protein by ribosomes. So yes, promoter is required to make mRNA. That's my understanding. DNA replication is done by DNA polymerases and other proteins I believe and does not involve mRNA. It may involve RNA in retroviruses but that's way beyond of my knowledge.



#153560 Are two promoters needed for co-expressing 2 proteins from the same plasmid in S

Posted by donny on 10 April 2013 - 06:52 PM in Molecular Biology

I have cloned two genes in series using one promoter but with an RBS before each gene for co-expression in E. coli and it worked. I've been reading up a little and from what I understand, prokaryotic mRNAs can be polycistronic while eukaryotic mRNAs are only monocistronic. So, many proteins can be translated from a single mRNA in E.coli but only one protein is encoded in every mRNA in yeast.

The promoter sequence controls transcription, not translation. Translation is performed by ribosomes. It binds to the RBS on the mRNA and stops at the stop codon in the RNA. Transcription is performed by polymerases and stops at the termination sequence in the DNA. I hope I got that right. I'm more a chemist learning molecular biology on the fly.



#153459 Are two promoters needed for co-expressing 2 proteins from the same plasmid in S

Posted by donny on 08 April 2013 - 08:56 PM in Molecular Biology

I'm working on Saccharmoyces cerevisiae for the first time and am intending to co-express 2 proteins from one plasmid. I have initially thought I can simply clone the genes serially and they will express under the same promoter, like in E. coli. Would this work in S. cerevisiae? I'm asking because I've read many papers and they all use pESC vector such that each gene is under the control of their own promoter. Is this essential?



#142865 Increasing cell growth rate in plain M9 medium

Posted by donny on 05 October 2012 - 10:54 AM in Microbiology

I have been trying to grow E. coli DH1 transformed with various plasmids in "plain" M9 medium, ie. this recipe with thiamine and NO other supplements like casamino acid, iron, trace metals etc. I've adapted the cells by subcultivation 3x but the growth rate is still significantly slower than rich media like LB. Adding casamino acid definitely increased growth rate but I don't want to use it because I want a real defined media. Are there other ways to increase the growth rate? Would adding iron and/or trace metals help?




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