Genotype meaning
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2.6 years ago
A_heath ▴ 160

Hi all,

I would like to have some help please to understand the genotype of a genetically modified bacterial strain (E. coli BL21 DE3) : F- ompT hsdSB (rB-mB-) gal dcm rne131 (DE3)

F- is for cells lacking the fertility factor but I don't have the other ones.

Thanks for your much appreciated help!

Audrey

genotype BL21 • 1.2k views
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F-: This strain does not carry the F plasmid (DNA plasmid called Fertility Factor or Sex Factor). 

ompT: Mutation in outer-membrane protease that Improves the yield of some recombinant proteins. 

hsdSB(rB- mB-): Allows cloning of DNA without cleavage by endogenous restriction endonucleases. 

gal: Mutation in galactose metabolism. Blocks galactose utilization. 

dcm: Cytosine methylation blocked (it makes DNA susceptible to cleavage at CC(A/T)GG sequences by some restriction enzymes). 

rne131: The rne gene encodes the RNase E enzyme, an essential, 1061 amino acid E. coli endonuclease which is involved in rRNA maturation and mRNA degradation as a component of a protein complex known as a “degradosome”

DE3: The DE3 designation indicate the strains contain the DE3 lysogen that carries the gene for T7 RNA polymerase under control of the lacUV5 promoter. IPTG is required to induce expression of the T7 RNA polymerase. 

Please find the details here and here.

Thank you.

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Thank you so much for this detailed genotype!

I have an additional question as I still identify lon proteases within the genome of BL21 DE3. Do you have any idea how is that possible as it is supposed to lack lon gene ?

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lon: This strain is mutant for lon protease (ATP-dependent serine protease that mediates the selective degradation of mutant and abnormal proteins as well as certain short-lived regulatory proteins, including some antitoxins). 

I still identify lon proteases within the genome of BL21 DE3

How did you identify lon proteases?

Did you sequence the BL21 DE3 from your lab or you are using the database sequence?

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On the genome of E. coli BL21 DE3 available on the NCBI database (CP001509.3), there is still at least one Lon protease (427,922 - 430,276 bp) and an additional one at: 1,021,628 - 1,023,388 bp so it doesn't make sense ... Thanks again for your help MSRS!

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Lon protease deficiency due to IS4 insertion in the promoter region of lon is a well-known genetic feature of E. coli B Reference

The IS186 at positions (426481..427823) of CP001509.3 play a role in the inactivation of lon protease deficiency. details here

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So much more understandable now... Thank you very much for all the help provided, it's greatly appreciated! Have a nice day,

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