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I need to regulate cAMP levels in E. coli in a very specific way- I need to produce excessive amounts of a phosphodieterase. To do that I need to clone a specific gene (cpdA gene) that is already in E. coli and then insert many more copies of it into each cell. Does anyone have any resources about specifically isolating a gene in E. coli and cloning it?
Thank you
cAMP regulation
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K.Kwan
- Posts: 9
- Joined: Thu Sep 08, 2005 9:28 pm
Here is one method that I found online, hopefully it can help you in some way
http://aem.asm.org/cgi/content/full/69/1/504
"To isolate the pdeA gene, genomic DNA was digested with ClaI and separated on an agarose gel. The 7-kb region of digested DNA was gel extracted and ligated into pBluescript cut with the same enzyme. The partial library was transformed into E. coli DH10B, and the transformed E. coli was plated onto LB agar containing 100 µg of ampicillin/ml and 32 µg of X-Gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside)/ml. White colonies were plated in a grid onto fresh LB agar plates, grown overnight, and replica plated onto a nylon membrane (Nytran N; Schleicher and Schuell). The cells were lysed by soaking the filter in a solution containing 0.5 N NaOH and 1.5 M NaCl and then neutralized with 1 M Tris-HCl (pH
. The DNA was cross-linked to the membrane by baking at 80°C for 1 h. Cell debris was removed by soaking the filter in 200 µg of proteinase K/ml dissolved in 1x TBS (137 mM NaCl, 25 mM Tris base, and 2.7 mM KCl adjusted to pH 7.4). The membranes were hybridized using essentially the same procedures outlined for Southern blots. The positive clone pSKT1 was sent for nucleotide sequencing by Genemed Inc. (South San Francisco, Calif.) and the DNA Sequencing Facility at the University of California at Berkeley."
http://aem.asm.org/cgi/content/full/69/1/504
"To isolate the pdeA gene, genomic DNA was digested with ClaI and separated on an agarose gel. The 7-kb region of digested DNA was gel extracted and ligated into pBluescript cut with the same enzyme. The partial library was transformed into E. coli DH10B, and the transformed E. coli was plated onto LB agar containing 100 µg of ampicillin/ml and 32 µg of X-Gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside)/ml. White colonies were plated in a grid onto fresh LB agar plates, grown overnight, and replica plated onto a nylon membrane (Nytran N; Schleicher and Schuell). The cells were lysed by soaking the filter in a solution containing 0.5 N NaOH and 1.5 M NaCl and then neutralized with 1 M Tris-HCl (pH
. The DNA was cross-linked to the membrane by baking at 80°C for 1 h. Cell debris was removed by soaking the filter in 200 µg of proteinase K/ml dissolved in 1x TBS (137 mM NaCl, 25 mM Tris base, and 2.7 mM KCl adjusted to pH 7.4). The membranes were hybridized using essentially the same procedures outlined for Southern blots. The positive clone pSKT1 was sent for nucleotide sequencing by Genemed Inc. (South San Francisco, Calif.) and the DNA Sequencing Facility at the University of California at Berkeley."-
carolinethorn
- Former Expert
- Posts: 393
- Joined: Tue Sep 20, 2005 2:40 pm
Hi mmb11,
What kind of facilities do you have for doing this experiment? In order to clone your gene of interest you would usually have to do a PCR reaction and then restriction digest to put the gene into a plasmid or vector. You would need to know the sequence of your gene of interest to design the PCR reaction to make copies of it. Then you would transfect the vector into your E.coli cells.
If you are not working in a lab with a PCR machine that would be tricky.
An alternative way could be to generate cAMP by stimulating with a drug that then activates adenylyl cyclase and generates cAMP.
Perhaps if you give some more details about your project I can be of more help.
-Caroline
What kind of facilities do you have for doing this experiment? In order to clone your gene of interest you would usually have to do a PCR reaction and then restriction digest to put the gene into a plasmid or vector. You would need to know the sequence of your gene of interest to design the PCR reaction to make copies of it. Then you would transfect the vector into your E.coli cells.
If you are not working in a lab with a PCR machine that would be tricky.
An alternative way could be to generate cAMP by stimulating with a drug that then activates adenylyl cyclase and generates cAMP.
Perhaps if you give some more details about your project I can be of more help.
-Caroline