Genetic Engineering

Genetic Engineering

  • Nina Odermatt added an answer:
    Is there any mycobacterial protein which is non toxic when over expressed in M.tuberculosis but toxic when over expressed in M.smegmatis?

    Hi, I am characterizing the transcriptional regulator of Mycobacteirum tuberculosis .

    Because we don't have facility for M.tuberculosis culture, I am trying to over express (using PVV16 which has Hsp60 promoter) it in M.smegmatis. but I never got colonies though the clone and vector back bone are fine. So I thought over expression might be toxic to M.smegmatis. so to check its toxicity I did frame shift mutations and found its over expression is not lethal.

    However for the same protein Chip-seq data available in MTB Network Portal. By going through their publication in NAR, I realised they over expressed corresponding transcription factors in M.tuberculosis using Tet-inducible system and did Chip-seq analysis.

    My question is, is over expression of my protein not toxic to M.tuberculosis but toxic to M.smegmatis or am I simply doing something wrong?

    How can I get a over expression strain of M.smegmatis?

    Nina Odermatt · École Polytechnique Fédérale de Lausanne

    Hello,

    There could be any reason why it doesn't work. How do you check the overexpression, by western blot?

    Is the protein of Mtb also present in M. smegmatis? If yes it could be toxic when already overexpressed in a little amount, as Msmeg already has a basal level of the protein. But even if the protein is ectopically expressed in Msmeg, maybe the Hsp60 promoter is just too strong, so that an overexpression can be toxic, but it is not with the inducible tet system (where you can adjust the expression and induce only slight overexpression to see an effect but not kill the bacteria).

    Best

  • Sanjeev Kumar added an answer:
    How to assess genetic stability within generations of clonaly propagated plants using molecular markers?

    I am following a research paper which is published from our research group.I am enclosing the paper with the question. I want improvise the data analysis part of the work which I intend to do. Apart from just assessing the somaclonal variations what additional approach can be incorporated? Any constructive suggestion is most welcome.

    Sanjeev Kumar · Indian Institute of Sugarcane Research

    For genetic fidelity testing one can use markers like SSR or ISSR ..but make sure that the sample size as well as the number of primers tested are sufficiently large.  Always use parental DNA and totally unrelated or even DNA from other source as  controls. The data will tell you two things.... Presence of any variant........and also true to the type plants (if you intended for that)........

  • Rong Ye added an answer:
    Does anyone have any advice on pET28a expression vector?

    I transformed the plasmid into DH5α and obtained about 30 colonies which i set out to verifiy. Ii selected 10 of them and 8 tested positive for KAN gene by PCR. However, upon subculturing the individual clones into LB broth supplemented with 40 ug/ml KAN, they did not grow up. Incidentaly, the same clones grew up on KAN-containing plate of same concentration. I then used the subcultured colonies from the KAN-containing plates to subculture in LB broth without any KAN. Upon extraction, no bands were observed on gel, but the concentration was 350 ng/ul by the Nanodrop machine. What could be the problem?

    Rong Ye · Fudan University

    I think your PCR results were be interfered.

  • Catarina A. Marques added an answer:
    What does it mean when a gene cannot be knocked out?

    Efforts to knock out the msp-2 gene in plasmodium have been unsuccessful. What does that mean with respect to the gene?

    Catarina A. Marques · University of Glasgow

    Hello, Plasmodium is haploid, so only one allele to KO. I guess if several strategies are employed and you can't ever get a KO it might suggest that the gene is essential. The problem is that it is never the most convincing way of saying "it is essential", you are never 100% sure if the parasite can't live without the gene or the transfection rate is too low, or something went wrong with it. And the problem in Plasmodium is that you transfect the parasites in the blood stage, and an essential gene in that stage might not be so in the other stages. I would look at papers that have been successful or attempted KO genes for more info. Also, I think that there are some groups working on conditional KO systems, I would take a look at them as well. Good luck.

  • Pascal Leclair added an answer:
    Does anyone know how long after plasmid transfection does it take for the CRISPR/Cas system to knock down the target gene?
    My target cell is 293T.
    Pascal Leclair · Child & Family Research Institute

    I think it depends on the gene (and maybe the cell line...). Using Jurkat cells, I had complete knock-down of one gene after about 7 days, a different gene took about 2 weeks.

  • Arvind Singh Chandel added an answer:
    Why am I not getting insert amplification after few days of cloning in a pCAMBIA vector?

    I cloned my gene (474 bp) in the pCAMBIA plant expression vector and transformed in DH5 alpha strain. After colony PCR I took the positive colonies for plasmid isolation and kept the isolated plasmid at -20 DC temp.

    But after one week when I again amplified the plasmid and positive colonies with gene-specific primers, it came back negative.

    What could be the probable reason behind it? How can all positive colonies become negative? I rechecked it many times with a positive control but also I did not get any amplification.

    Arvind Singh Chandel · Central Salt and Marine Chemicals Research Institute

    Yes Maureen J Ostaff.... you are right , she should check once her construct through RE double digestion and confirm that insert present in gel or not. after that trouble shoot trough PCR components. 

  • Andrey Sinjushin added an answer:
    Are the results that are obtained from AFLP reproducible?

    AFLP requires a high quality DNA, right estimation of DNA quantity, and professional person has not any mistake or the error should not exceed 5 %, therefore my question is: Are the results that are obtained from AFLP reproducible?

    Andrey Sinjushin · Lomonosov Moscow State University

    They are said to be reproducible, yes. However, you need to make a positive control (gel lane with PCR product obtained from PCR mix without DNA - it should be free from DNA bands). If you want to test the reproducibility, you surely may remake all the reactions with one sample, then check whether the lanes reproduce and conclude that everything is ok. I think it is not neccessary.

  • Roni Hogri added an answer:
    Difference between localization of gene expression in AAV Vs. lentiviral vectors?

    Dear community,

    We use viral vectors to deliver genes in optogenetic experiments in rats. I have recently read a claim that lentiviral vectors result in more localized expression as compared to AAV (e.g., Yizhar et al, Neuron 2011; Hirai, Cerebellum 2008). We usually work with AAVs as it is more simple (especially with regard to safety protocols).

    1. What is your experience with this issue - did you observe such differences in your preparations?

    2. If this is indeed the case, does anyone have an idea of the mechanism underlying this difference in the level of localization?

    Thanks,

    Roni

    Roni Hogri · Tel Aviv University

    Yes, I deliver the virus stereotaxically.

    Thanks for the paper, it should be quite helpful!

  • Yamini Shah added an answer:
    What is the best percentage of glycerol stock preparation for transformed culture recommended for the long term storage?
    What is the best percentage of glycerol stock preparation for transformed culture is recommended for the long term storage? How long will cultures be efficiency maintained?
    Yamini Shah · Reliance Industries Limited

    40% Sterlised Glycerol  is ideal & used as  1:1 (sample : 40% Glycerol) ratio  with generally log phase pure bacterial culture cells grown in LB/NB/TYG.

  • Shahin Eghbalsaied added an answer:
    Does anyone know if there are any IRES-like sequences in prokaryotes that work?
    Longer Explanation: I would like to express a fusion of protein X and Y in prokaryotes. However, I want Y to be expressed by itself as well (from the same promoter). In a mammalian system (which I am more familiar with) I would just simply put a read-through IRES in between X and Y. This would result in the gene products XY as well as Y alone (ratio depending on how good the used IRES is). An alternative would be to put the FMDV 2A translational skip peptide in between X and Y which would yield the gene products XY and X + Y (ratio depending on the efficiency of 2A). However, I don't know of any IRES that can be used in prokaryotes and also the FMDV 2A peptide is not active in prokaryotes. I was also thinking if a protease site in between X and Y would work (if it is only partially efficient). Also, one could consider a read-through stop-codon (amber) but that would result in XY and X rather than XY and Y (the fusion must be XY and not YX). Anyway, if any of you good prokaryote scientists out there have any suggestions for an eukaryotic scientist, please help! Thanks!!
    Shahin Eghbalsaied · Islamic Azad University Khorasgan (Isfahan) Branch

    Hi Daniel,

    How was your experience with prokaryotic RBS in terms of expression for the second transgene? What about the fused protein?

    Thanks.

  • Miloslav Kitner added an answer:
    Should I do AFLP restriction and ligation together or not?

    In my protocol for AFLP, I'm doing first restriction for 4.5 hours, then ligation that requires at least 9 hours and therefore the cycler works overnight. Recently, in some protocols, one can do restriction and ligation together. What is more efficient, doing every step separate or together?  

  • Olivia Zabel added an answer:
    What is the simplest and cheapest method to transfer a gene to mammalian cells ?
    I want to transfer the GFP protein gene to my cell lines. I want know the simplest way to do so. Preferably a chemical method.
    Olivia Zabel · Lipocalyx Gmbh

    You may use Viromers (from Lipocalyx). I am working with Viromers too and I am very satisfied with transfection efficiency. Viromers are synthetic polymers and zero in charge. Due to their endosome escape mechanism I achieve higher transfection efficiency compared to standard transection reagents.

  • Rama Kant Dubey added an answer:
    What is transgene pollution and how is it related to horizontal gene transfer?

    Over-exploitation of genetic engineering and inter specific gene transfers into the genome.

    Rama Kant Dubey · Institute of Environment & Sustainable Development,BHU, Varanasi,U.P., India

    Dear Namrata

    Its a problem arises with the movement  of gene/gene segment between two organism or plants. it is kind of limitation in the transgenic technology.Mostly microbial based gene are playing more important role in the horizontal gene transfer. Developing resistance in the microbial strains is also a reason of HGT.

  • Millie Lam added an answer:
    Why would one get a low fold induction upon doxycycline treatment?

    Has anyone used the FUW-tetO system for doxycycline-induced transgene expression? I transduced my cells with trangenes (MOI=5 each) under the FUW-tetO, then treated with 1 ug/mL doxycycline for 5 days. The fold induction was only like 1.5X to 5 X which seems very low when checked by real time PCR

    Is it normal for this system to give such low fold induction? or has anyone got a much better fold induction using this system?

    Thanks

    Millie Lam · National University of Singapore

    Thanks a lot! I am going to use Tet-free FBS for my subsequent experiments!

  • Margaret Mchenney added an answer:
    What would be the most appropriate nucleotide substitution model(s) for GC-rich genomes of actinobacteria?

    Typical GC content is around 70 % (Streptomyces). To my knowledge, there is no reliable experimental data on frequencies of different kinds of mutations in this group (except Mycobacterium tuberculosis, but I am not sure as to whether we can extrapolate Mtb data on free-living strains, or can we?). What would the null hypothesis be in this case? Can we gain necessary information through comparison of related genes/genomes?

    Margaret Mchenney · Eli Lilly

    I reject the null hypothesis in this case.  My best guess is that one could potentially extrapolate Mtb data on free living strains and could potentially gain interesting information through comparison of related gene/genomes.  Why don't you try a BLAST analysis of the genes that you are interested in comparing and see what you come up with.  Look for conserved sequences.

  • Alina Filatova added an answer:
    What are some efficient methods to clone multiple guide RNAs into a CRISPR/Cas9 nickase expression vector?

    What are some efficient methods to clone multiple guide RNAs into a CRISPR/Cas9 expression vector like pX462? How can they be optimally implemented in practice? 

    I was considering the "Golden Gate" technique but I have no experience with this. Anyone have some advice for the multiplexed CRISPR wannabes out there?

    Here is a link to the plasmid: http://www.addgene.org/48141/

    It has ampicillin, puromycin resistance and BsbI cleavage sites. 

    Alina Filatova · Justus-Liebig-Universität Gießen

    Hi Salvatore,

    Could you please describe in more detail the cloning procedure? How did you design the oligos? Did gRNA contain both sgRNAs for double nicking?

    Thanks a lot!

  • Anchel Gonzalez added an answer:
    What is the best linker for a fusion protein?

    Hello everyone, I am preparing a fusion protein with mCherry and I have doubts about the type of linker to use. I have seen in the literature that sometimes flexible linkers of this type of sequence are used: (Gly-Gly-Gly-Gly-Ser)n. Can an expert in the field confirm whether this residue composition is the most suitable? What would be the optimal length?

    Thank you for your help.

    Anchel Gonzalez · Radboud University Medical Centre (Radboudumc)

    Thank you all for your answers, I will try this linker first: Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly

    And let's see how it goes!

    Thanks!

  • Mohammed Yaro added an answer:
    Will the PCR of my DNA be affected if I do not follow the correct steps for DNA rehydration, as outlined by my kit instructions?

    I did a DNA extraction, but I am not sure if the rehydration step was good or not.

    The kit instructions told me to keep the DNA at 60°C for 90 minutes, but I only kept it there for 30 minutes.

    Mohammed Yaro · Biotechnology and Nuclear Agriculture Research Institute

    You could also check the concentration using a nanodrop it provides quick result. Generally speaking, i will advice you stick to the protocol as indicated by the manufacturer, because usually a lot of  due diligence goes into it.

  • János Zsámboki added an answer:
    Has Anyone successfully cloned a 1.5 Kb insert on a 7.5Kb vector with just one sticky end and a blunt end on the other side on both?

    We've digested both insert (from purified pcr product) and vector (from maxi prep) with BamH1 ( leaving 1 sticky end) and with HPaI (leaving a blunt end). We have tried the ligation several times but we were unsuccessful. Before we change the approach I was wondering if anyone has any idea why this is not being possible. Thank you!

    János Zsámboki · Hungarian Academy of Sciences

    Another thing that popped into my mind: You can add PEG to the reaction to increase macromolecular crowding to facilitate the ligation of the blunt end.

  • Koen Venken added an answer:
    I want to make a Gal4 transgenic zebrafish line and need a way to follow germline transmission by fusing mCherry to Gal4, will this disrupt activity?

    Gal4 zebrafish transgenesis. How to identify germline carriers.

    Koen Venken · Baylor College of Medicine

    You can use the 2A self cleaving peptide polycistronic strategy.  Put a 2A peptide (e.g., T2A) between mCherry (without stop codon) and GAL4. It will make a 1:1 Cherry and GAL4 in each cell where the GAL4 is expressed.

  • Sarwan Kumar Dubey added an answer:
    Can some one tell the rice varieties specially bred for Aerobic conditions or one can say Aerobic rice varieties?

    Most of the time varieties bred for wet lands are normally being used for aerobic and/or DSR conditions. I am interested in varieties specially developed to grow in Aerobic condition in semi arid part of India.Thanks.

    Sarwan Kumar Dubey · Central Soil and Water Conservation Research and Training Institute

    Thanks Dr Ghislain Kanfany; I am in touch with Dr RK Singh. Regards

  • Chunxin Wang added an answer:
    Are there general protocols that can be applied in most cases on how to carry out the optimization of sgRNA for CRISPR/cas9 method?

    It is known that the CRISPR method can have off-target effects if this is not well addressed by optimizing the sgRNA, taking into account that CRISPR has been used in genetic engineering from relatively recently, are there currently general "rules" for the sgRNA editing?

    Chunxin Wang · National Institutes of Health

    There is one paper reported that using certain chemical to help loosen the chromatin structures may enhance TALEN targeting. Some also argued that methylation may affect targeting efficiency as well for TALEN. I'm no aware of any similar reports with CRISPR. But since CRISPR is generally very effective, you really don't need to worry too much. Construction of CRISPR is so easy that you can just try several different gRNAs targeting different regions to find the efficient one. 

  • Elma Mrehic added an answer:
    In DNA extraction from saliva, why doesn't the pallet dissolve in the tube after adding DDH2O?

    I am extracting DNA from Saliva through Manual Protocol NOT with the Standardized Kits. Right at the end of the extraction when I get the DNA Pallet that need to be dissolved by adding de-ionized water which normally does dissolves and we process it to further step gel electrophoresis. But I am facing difficulty in this step that is the pallet which does not dissolved. I am using Phenol, Chloroform, EDTA, Tris, BME and PK and Lysis buffer. Some of my colleagues ask me to wash the Phenol with TAE Buffer solution this will help you in proper extraction. Kindly help me out in this regard.

    Elma Mrehic · International BURCH University

    1X Tris EDTA buffer and leave the samples at 4 ° C overnight

  • Seeds DNA extraction?
    Which protocol is the best for extracting seeds DNA contents? With considering seeds of the poppy plant being used.
    Ketankumar Joitaram Panchal · Sardar Patel University

    You can try kits as well as conventional protocol but keep it in mind that what plants species you are targeting because poyphenol content will vary with plants species

  • Noha Said added an answer:
    What is the difference between PCR-tetra primer ARMS, PCR-AS, and PCR-CTPP?

    I want to know the difference between PCR-tetra primer ARMS, PCR-AS, and PCR-CTPP. I want to use the most simple one in the genotyping.

    Noha Said · Zagazig University

    THANK YOU

  • Rolf Henrik Nilsson added an answer:
    Is there any software or online tool to check the purity of gene sequence?
    How can we check the purity of the gene sequence (in percent)? Is there any software or online tool or any calculation?
    Rolf Henrik Nilsson · University of Gothenburg

    Some ideas on how to examine sequences for quality and reliability are presented here:

    https://www.researchgate.net/publication/235931750_Five_simple_guidelines_for_establishing_basic_authenticity_and_reliability_of_newly_generated_fungal_ITS_sequences

    https://www.researchgate.net/publication/235601668_Incorporating_molecular_data_in_fungal_systematics_a_guide_for_aspiringresearchers

  • Aldwin Anterola added an answer:
    Does anybody have whole sequence information of pART7 vector?
    I need whole sequence information of pART7 vector.
    Aldwin Anterola · Southern Illinois University Carbondale

    I found the sequence here:

    http://www.csiro.au/Organisation-Structure/Divisions/Plant-Industry/RNAi/Plant-vectors.aspx

  • Pradeep Kumar Brahman added an answer:
    What should be the DNA absorbance ratio so that it can be used in Genetic Engineering? (during purification of DNA)

    DNA absorbance ratio

    Pradeep Kumar Brahman · Koneru Lakshmaiah University

    Actually question is nor clear. In what kind of work that ratio is needed. If you run the UV spectrum of ds-DNA it gives absorption peak at 260nm. However if you clarify your applications I can help. Because I am also working on DNA analysis.

  • Chantal Agbemabiese added an answer:
    Why do my sequences have low signals at the 3 prime ends?
    I do a pre sequencing purification using EXOSAP-IT and I sequence using the Big Dye terminator cycle sequencing kit v.3.1. I then clean up using Sephadex G50 on a multi-screen filter plate before sequencing.
    Chantal Agbemabiese · Nagasaki University

    Thank you Pia. I was able to optimize my reactions finally.

  • Yuan-Yeu Yau added an answer:
    Can splicing occur when a bacterial gene is transfected in an eukaryotic system?

    Will splicing occur in a countinuous gene expressed in a eukaryotic system, if so, what steps can one take to prevent such potential problems?

    Yuan-Yeu Yau · Northeastern State University

    Most likely, if the bacterial gene sequence contains consensus sequences for an intron to be recognized by the splicing machinery in the eukaryotic organism.

    GUS gene is from bacteria. Researchers had inserted a plant intron into the gus gene and transferred into plants, and found out that the intron was efficiently spliced and gave rise to the GUS enzymatic activity (see attached paper).

    We had tried to study the expressing ability of several site-specific recombinase (SSR) genes from prokaryotes in higher plants. We usually scan those intron consensus sequences in the genes to predict the possible outcome. As John suggested, one can mutate out those intron motifs with site directed mutagenesis to avoid unwanted splicing.

About Genetic Engineering

Directed modification of the gene complement of a living organism by such techniques as altering the DNA, substituting genetic material by means of a virus, transplanting whole nuclei, transplanting cell hybrids, etc.

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