[ccp4bb] OFFTOPIC question "Two plasmids in one host cell"
Hi All, I have two constructs having different ori, p15ori and M13 ori, different promoters araBAD promoter and LacI, and different antibiotic resistance chloramphenicol and Ampicillin respectively. I am managed to get the transformants and getting the expected result after blunt digestion with the EcoRV enzyme. Since both the plasmids have the site for EcoRV. But the p15 ori has a low copy number that's why I am seeing the very faint band as compared to other plasmid in the sample. So I would like to know is there any way that I can quantify the low copy number plasmid. Because I am not able to sequence it with the specific primers it could be due to its low concentration. Please advise. Thank you. -- Dr. Anamika Singh Post-Doctoral Fellow Silberman Institute of Life Sciences Hebrew University of Jerusalem, Israel No: 054-294-8036 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] OFF TOPIC question
*One correction to the previous question:* I have two constructs having different ori, p15ori and M13 ori, different promoters *araBAD promoter* and LacI, and different antibiotic resistance chloramphenicol and Ampicillin respectively. I would like to know which E. coli host cells will be good for the co-transformation of these constructs? On Thu, 26 Nov 2020 at 11:59, Anamika Singh wrote: > Hi all, > > I have two constructs having different ori, p15ori and M13 ori, different > promoters araC and LacI, and different antibiotic resistance > chloramphenicol and Ampicillin respectively. I would like to know which > *expressing > E. coli host cells* will be good for the co-transformation of these > constructs? > > Thanks > > Anamika > > -- Dr. Anamika Singh Post-Doctoral Fellow Silberman Institute of Life Sciences Hebrew University of Jerusalem, Israel No: 054-294-8036 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
[ccp4bb] OFF TOPIC question
Hi all, I have two constructs having different ori, p15ori and M13 ori, different promoters araC and LacI, and different antibiotic resistance chloramphenicol and Ampicillin respectively. I would like to know which *expressing E. coli host cells* will be good for the co-transformation of these constructs? Thanks Anamika To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
[ccp4bb] OFF topic Question Nucleotide exchange of RAS protein?
Hi All, Does anyone know what is the actual difference between CIP and EDTA method for exchanging the GppNHp (non-hydrolysable GTP) or GDP with the purified RAS protein? I have a protocol according to which I need to incubate the purified RAS with GppNHp having 20U of alkaline phosphatase, 10uM of Zinc Chloride and 200mM ammonium sulfate for 2 hr at room temperature. In this protocol, they have mentioned that ammonium sulfate needs to be added in the last. I am quite confused, according to them do I need to add it after 2 hrs incubation or I have to add this at last to the mixture of (purified RAS+GppNHp+Zinc chloride and alkaline phosphatase)? Since this protein and its mechanism is new to me so not sure what to do. After reading some papers also I am not able to understand which method is good for exchange and how these reactions behave? Please help me in this regard. -- Anamika Singh Post-Doctoral Fellow Silberman Institute of Life Sciences Hebrew University of Jerusalem, Israel No: 054-294-8036 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
[ccp4bb] POST_DOCTORATE POSITION
Hi All, On behalf of Julia Shifman, I am posting this advertisement. Shifman lab in the Hebrew University of Jerusalem is looking for a postdoctoral scholar in Experimental Protein Engineering. In our lab, we engineer inhibitors of protein-protein interactions starting from natural binding partners as well as from unrelated small protein domains. Such inhibitors could be evolved to selectively target only one family member among hundreds of homologs, making them attractive drug candidates. Using a combination of computational and directed evolution approaches, we convert the natural low-affinity binding partners into high-affinity and high-specificity binders. In the particular project, we would like to engineer specific inhibitors to various types of Matrix Metalloproteinases, enzymes whose activity is related to various diseases including cancer. These molecules will be later transformed into drug candidates. We are looking for a highly motivated person with a background in Biophysics or Biochemistry to take over this exciting project. The candidate should have experience with molecular biology and protein expression/purification. Experience in yeast surface display is a plus. Our lab is located in the Department of Biological Chemistry in the Hebrew University of Jerusalem, Jerusalem, Israel. Please contact Julia Shifman: jshif...@mail.huji.ac.il -- Dr. Anamika Singh Post-Doctoral Fellow Silberman Institute of Life Sciences Hebrew University of Jerusalem, Israel No: 054-294-8036 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
[ccp4bb] OFF TOPIC
Hi All, I am purifying the biotinylated protein (cloned into the pET28a vector) using Avidin beads. Since I need the protein for SPR but when I used the purified protein to interact with Streptavidin coated onto the SPR chip. There was no signal. Can anybody tell me why is it so or how can I make sure that the purified protein is biotinylated enough to interact and give the signal? Because when I ran the SDS-PAGE there was a band of purified protein. I have used the elution buffer (20mM Tris, 5mM Mgcl2, 500mM Nacl, and 2mM Biotin). PS: I have included the biotin during overexpression of the protein also. Please suggest. Thanks -- Anamika Post-Doctoral Fellow Silberman Institute of Life Sciences Hebrew University of Jerusalem, Israel To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
[ccp4bb] OFF topic (Protein degrades during Dailysis)
Dear All, I am purifying the His-tagged proteins ( 21Kda) using buffer 20mM Tris, 150mM Nacl, and 5mM MgCl2 with 500mM imidazole. Earlier I was facing the precipitation problem during dialysis but with the addition of 50mM L-Arg somehow managed to overcome the precipitation issue. But this time I have seen after overnight dialysis there are degradation products on SDS page. I have used protease inhibitor (PMSF) in my sonication buffer. Please suggest me to overcome this problem. Thanks -- Anamika
[ccp4bb] off topic
Hi, Is anyone has worked with STAT1 proteins? I have cloned the SH2 domain of STAT1 protein into pet28a vector but there was no expression so far or rather say inconsistent expression. Sometimes the expression was in inclusion bodies. I have tried different methods to pull out the protein from inclusion bodies using urea, guanidium chloride tween20 but none of them worked well. The yield was very low (very faint band on SDS-PAGE ) from 3-liter culture. I changed the host cells from BL21 to Rosetta DE3 cells but no success so far. We thought to use some other vector system like with SUMO tag but did not proceed because the aim of the project to design inhibitor and tag will interfere. Please suggest me something so that I can complete my project in lesser time. Looking forward to valuable suggestions. Thanks Anamika
[ccp4bb] About crystallization diffraction problem
Dear All, I want to get some help regarding crystallization for one of the protein I am working. This is a recombinant protein of molecular weight of 17.5 kDa. I am getting crystals shape like thin plates in 5 days, in different conditions like bis tris pH 6.5-8, HEPES pH 6-8 with .1M nacl , .01M DTT having PEG 3350, PEG 6000 as precipitant. But whenever we used to put crystal in cryoprotectant like 20 % ethylene glycol, glycerol, MPD it used to split like layers of some tree barks. And the crystal which were diffracting not getting diffracted above 3.0 Angstrom. Please help me out. Thanks in advance -- Anamika
