- Rajendra Angara 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?
Dear @Jan van Ooyen , thank you for your kind reply.
using Acetamide inducible promoter, i am able to over express the protein.Following
- John Laudenslager added an answer:Can stop codons stop translation when placed before the start codon of a protein?
If I introduce 3 Stop Codons (In the 3 different reading frames) just before the start codon of a genome protein without promoter will this stop the translation of my protein of interest in case its transcription is activated by a promoter upstream?
Depending on the detail of your plan, you could simply try removing the ATG from your GOI, making sure you don't have in-frame upstream ATGs in your cassette and checking for other downstream in-frame ATGs in your GOI that may still produce a "functional" protein. In that case your GOI should not be translated to a functional protein. The subsequent recombination event could then introduce an in-frame ATG along with the promoter. But I don't know the exact plan/goal so that approach may no be feasible or desirable.Following
- Zeeshan Chaudhry added an answer:How can I add a GFP to a specific gene?
I want to add a GFP to a specific gene to trace the gene expression and for the flow analysis. I hope that the GFP can be cut when translated so that GFP wont affect the function of the gene. what should I do? Can some one give me some related papers?
I am not 100% sure if you want to track the gene expression with GFP for Flow analysis. Under what kind of promoter your gene would be ? its natural promoter or a high throughput like CMV promoter. If it would be under its natural promoter then it would definitely be regulated. Be careful if the expression of the gene is enough to generate proper amount for the Flow cell to detect the signal. Although they are sensitive enough but sometime it might be a problem.
Now coming on to how to do this. Fusing the GFP might not be a big problem just for the expression detection. But if you want a functional copy of your protein as well then there are options like he 2A peptides and IRES (internal Ribosomal Entry Site). Theoretically the 2A peptides are better as they give eqimolar concentration for both cistrons. But I have worked with only the 2A peptides and not IRES so I cant say which one is better.
If you could provide a little more detail as to what do you plan to do.Following
- Tuhin Kumar Guha added an answer:What can I do with the MBP fusion protein expression that does not work?
One of the proteins (homing endonuclease) which I am trying to express and purify has been giving a hard time since.
I have cloned and sequence checked the ORF and its in frame with the MBP (cloned in pMALc5x from NEB), everytime when I try to express this ORF using various IPTG conc., temperatures eyc. it does not. However the empty control pMALc5x expresses very nice when i induce with 0.3 mM IPTG.
The full length protei is never achieved...I am thinking of various pointers like changing the cell strain, doing western, media etc. but I would also appreciate inputs from those who have experienced such problem.
Any suggestion will be appreciated and acknowledged.
Thanks in advance
@Rakesh Kumar : Few years back I tried expressing my protein in a similar manner which you have indicated but did not work with the fusion one though. I will keep your pointers in mind.
@ Pierre Béguin: I guess so. Proteolysis may be the problem. I am trying out various other lower temps. to see whether that makes a difference or not. I have tried his tag but the protein was unable to purify. I appreciate you for the pointers.
@Zeeshan Chaudhry: That will be beyond the scope for the moment, however I thank you for the reply.
@Anna Leopold: Thanks for the reply.I have been doing small scale protein expression to optimize the expression of this fused protein. the prdicetd molecular weight of the fusion protein is 74 Kda (Maltose binding protein is 42 KDa and the recombinant protein is 32 KDa). Ya I agree with you but one time I expressed efficiently 110 KDa yeast peroxisomal protein in E.coli. Well, I tried to purify using his tag but something always goes wrong with that purification.I kinda switched to the pMAL expression system. Western does give the impression that the full length protein is formed at some point and there are few intermediate bands when detected with anti-MBP antibody. pET32 may be a good choice, I can think about that later.
@Hüseyin Besir: Thank you. I tried in vivo endonuclease assay where I co transformed both the expression construct and the substrate plasmid and did in vivo cutting assay. I found the cells were quite viable, so this is not a toxic protein. the sequence of the fusion protein is in frame and we checked it twice and did not observe any mutation whatsoever.
@Ian R Wilkinson: Thanks for the reply. Yes we got me right. The protein is not toxic, the cells are happy even after induction. Its under tight promoter ptac so that's not the problem. Currently, I am transforming my expression construct (in pMAL vector) with pRARE plasmid and hope to see some more full length expression. Time scale experiments and changing media had been done with no such great difference in the expression of the full length fusion protein.
I did not think about the repeat sequences, but we checked the in silico model of the protein, there are no such numerous repeat sequences. pMALp5x can express my fused protein in the periplasm if the protein expression is way too much affected with reducing environment or disulfide bond formation. I have that in mind.
@Christopher Wilson: Thanks for the reply. The insoluble fraction and the soluble fraction kind of look the same. SUMO approach ..lets see you far I can stretch this pMAL stuff.Following
- Weixun Li added an answer:Can someone recommend a software similar to Gene Runner, with such options as DNA translation in all frames, searching oligos, designing primers?I've got a windows 7 on my PC and can't install Gene Runner on it. I searched a lot but couldn't find a software which contains all the possibilities together. Does anyone have any suggestion?
New edition has released. After 3hrs trial, I think new one is way better than previous one. The developer really give me a surprise.Following
- Miguel Reina-Campos 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.
I am in the same situation. Thumbs-up for the linked Mol Cell paper, but If you want to use the Feng Zhang's constructs, here is the approach I am trying:
1-Clone one sgRNA into PX461
2-Clone second pair sgRNA into PX462
3-Transfect your cell of interest with PX462 and select with puromycin
4-Transfect your selected cells with PX461 and sort for GFP, one cell per well, in a 96 well plate. Try to use conditioned media supplemented with FBS and glutamine (o whatever your cells like) to collect your sorted cells.
Ideally I would like to replace GFP with RFP so I could clone one sgRNA in each construct, cotransfect and sort for "orange" cells. But the puromycin approach seems promising to me.
I will keep you posted.
- Umesh P added an answer:What is the difference between genetic engineering and synthetic biology?
Synthetic biology, synbio for short, is an emerging interdisciplinary field that in an essence attempts to bring engineering principles into biology. Nontheless the idea of engineering biological systems has been often confused with what has already been done in other areas of biotechnology such as metabolic engineering and genetic engineering, and this has been one of the major reasons of why synthetic biology is hard to define, even by leading experts in the field.
I know that the main difference between synbio and GE is the use of new foundational tools ( asides from those already used in Genetic engineering like recombinant DNA technology ) that have upgraded the potential of GE.This new tools such as DNA synthesis, the use of Standards and Abstraction have helped the development of synbio.(This idea is explained by Drew endy in these video :https://www.youtube.com/watch?v=XIuh7KDRzLk)
But here comes my real question: Is the use of these 3 foundational tools (standards,DNA synthesis ,abstraction) necessary for a project to be considered Synthetic biology?
In other words a project that contains the use of DNA Synthesis , but doesn't use standards (i.e. biobricks ..) or abstraction would be considered "genetic engineering" or synthetic biology?
i Personally think that the use of these 3 things is essential for it to be considered synbio , but i am no expert so i would like to know because I am writing an article for a school journal and i would like to explain it properly. thank you
Synthetic Biology is Extreme Genetic Engineering..
Synthetic Biology is rational design of genetic circuits by using modelling, analysis and Computer scienceFollowing
- Bassem Refaat added an answer:Does anyone have any advice about the best method for RNA isolation purity from paraffin blocks for RNAseq experiment?
Hi, I would like to ask about the best method to get high quality RNA to be used in RNAseq experiment from Paraffin blocks.
I would recommend using commercial kits either from Applied Biosystems (Ambion) or Qiagen. both kits works pretty well.
- Bhrugu J Yagnik added an answer:Which part of the plant is most preferred for DNA isolation?
Different parts of the plants can be used for the isolation of genomic DNA. But which part is most preferred for DNA isolation and why?
Young fresh leavesFollowing
- Rongfeng li added an answer:Any advice on how to make E.coli- S. aureus shuttle vector- to express GFP/any fluorescence protein?
I just want to get a shuttle vector (E.coli- S. aureus) expressing GFP or any fluoresence protein. If there is any plasmid as such, please sent me the details regarding the plasmid, its cost etc
you can find this kind expression vector from other labs. This would make your job much easier. But if you construct it by yourself, you need two replicons, one for replication in E. coli and the other for S. aureus. You also need antibiotic selection maker(s). Hopefully you can find a resistant gene that works in bother strains, otherwise, you have to put two in the vector. Finally, you need a strong promoter for expression in S. aureus and add MCS downstream to allow you to clone the GFP. You probably also need to put a terminator downstream of MCS. Good luck.Following
- Ganapati Bhat added an answer:Does anyone know the sequence for the multiple cloning site in Lonza's pmaxGFP or at least the order of restriction sites upstream of GFP?
Lonza provides a plasmid map of pmaxGFP showing the KpnI, NheI, Eco47III, and AgeI restriction sites upstream of max GFP and BglII, XhoI, and SacI downstream. Does anyone know if there are additional sites upstream of GFP? I would like to try and clone my GOI from another expression plasmid into this one due to its small size.
One more entry is in addgene please check and might be useful
- Josef Bober added an answer:Is it really tough to electroporate Lactobacillus reuteri?I am working with a plasmid called pSIP 411. I have tried several methods of transforming this plasmid DNA in to L. reuteri like high salt, glycine enriched, PEG, sucrose, high voltage etc. I certainly fail!
L. reuteri grows well in aerobic/ micro-aerobic conditions. Is it necessary to grow them anaerobically during the incubation of plates after electric shock or during competent cell preparation?
Is there any other method to transform Plasmid in to L. reuteri?
The latest thesis attached here says "Despite our efforts our L. reuteri strain remained reluctant to any transformation protocol which might be due to its pronounced agglutination and therefore inaccessible cell wall."
How can I overcome this agglutination problem?
We have many of the latest papers that have used electroporation in L. reuteri. How do they do this? Any ideas, please let me know.
Were you able to resolve this issue? I also have been having trouble transforming a similar plasmid into L. reuteri.Following
- Elissa K Leonard added an answer:What is a good vector mapping software?Can anyone suggest a suitable vector mapping software? Suppose I have a sequence of a vector, I want to know the promoter, terminator, antibiotic resistance etc. in a region. I used NEB cutter, but this one is useful only to track restriction sites and CDS.
I am a huge fan of Snapgene. (http://www.snapgene.com/) There is a free 30 day trial of the full version, but "Snapgene Viewer"--which has most of the functions you're looking for (automatically annotates various features, allows you to see/choose restriction enzymes, generates plasmid maps, and much more...)--is completely free. Full disclosure: I have not yet tried many of the alternatives, but that is mostly because I am so happy with Snapgene.Following
- Vikas kumar Patel added an answer:Can anyone suggest me a manual protocol for DNA purification from agarose gel?I am trying to elute a DNA band from agarose gel using the manual protocol; in this I cut the DNA band from the gel on the transilluminator (tried to expose to UV light for very short duration), put it in a syringe at -20 C for 30 minutes, pressed it tightly and collected the elute (water droplets from gel) in an eppendorf, purified it using the phenol-chloroform method and precipitated using NaCl and ethanol, washed with 95% ethanol.
I got the results but very poor yields (as visualized again on gel), not sufficient for cloning.
thanks to all of youFollowing
- Yanshan Liu added an answer:Is the usage of SSA and T7E1 assay in CRISPR/Cas9 or TALEN, necessary?
when you use CRISPR/Cas, TALEN, ZNF or other methods to manipulate genome, do you also do SSA or T7E1 assay to test if the system works? Is it necessary to do both or you choose one to test or it is totally waste of time and money to do this kind of testing?
I always use SSA assay with CRISPR, in which I cloned the target region into a reporter vector to test the system before I really do it in cells. And it worked every time. I think it is reasonable, 'cause you transfected the reporter into the cell, you have more than plenty of targets to bind, it doesn't mean that the it also can bind the endogenous targets. With T7E1 there is another kind of situation, you know the system works, but you are not sure whether it binds the real target or just because of off-target effect. Am I wrong, or what you usually do?
Thank you for your answers, guys. That helps a lot.Following
- Yuan-Yeu Yau added an answer:Why I have some tissue give me 200 bp only and others 200 and 500 bp in RT-PCR?
I transferred cDNA to the organism that should be have this gene in their genome. Just did transfer cDNA to increase the expression of this gene.
I did RT-PCR by specific primer for cDNA. According to NCBI this primer should give me a specific fragment for cDNA (200 bp) and other fragment for the genome (500 bp).
My result showed that non-transgenic have fragment size (500 bp) in different tissue.
Transgenic have fragment size 200 bp and 500 bp.
some tissue have only 200 bp and other tissue have both 200 bp and 500 bp.
To draw a concrete conclusion that "most of your transgenic showed only one band as positive and few of others showed two band", your need to be pretty sure that your PCR conditions can be used to amplify both fragments properly.
Nermeen, are you using ONE common pair of primers to try to amplify 2 fragments (one long: 500 bp and one short: 200 bp) in one PCR reaction?Following
- Sudhindra R Gadagkar added an answer:Does the rbcL gene contain introns?
It appears that the large subunit of the ribulose-bisphosphate carboxylase gene (rbcL) does not contain introns, at least not in the species I have checked (e.g., Camellia impressinervis; see http://www.ncbi.nlm.nih.gov/gene/17098326 and http://www.ncbi.nlm.nih.gov/nuccore/NC_022461.1?report=fasta&from=57095&to=58522). Does anybody know if it does contain introns in any species? If so, could you point me to the complete gene sequence with exon/intron boundaries, please? Thanks.
Thanks Prabodh and Humberto. Unfortunately, not seeing introns is not proof that this is an intronless gene. But thanks anyway.Following
- Raj Kumar added an answer:How can I select the transfected cell to make a stable cell line when cell and plasmid both are G418 (Geneticin) resistant?
Cell and plasmid both are G418 (Geneticin) resistance. So how can i select the transfected cell to make stable cell line. I need to work in same cell line and same plasmid. so any answer is appreciated. Thank you.
Thanks Dr Trevor Huyton that is the option left need to do.Following
- Joseph Miano added an answer:In CRISPR system, what is the concentration of each component to be used when microinjection?The best amount of gRNA vector, Cas9 mRNA, and ss donor DNA.
50 ng/ul sgRNA, 100 ng/ul oligo and 50 ng/ul Cas9 mRNA for startingFollowing
- Hamed Fakhim added an answer:What is the additional bandwidth in electrophoresis?
It is electrophoresis related to specific primer for Candida albicans ,from a patient with vaginitis.
In first row after ladder in sample 7, we distinguish two different pcr product close to Each other. we apply purify colony without any contamination.
hi dear javad
it seems that you work with HWP1 primers for separate c.albicans complex.
with this primers in some species of c.albicans we get additional band.
but it needs that we do more research about this, due to it happens for some species.Following
- Lorraine Clark added an answer:How can I produce recombinant ENZYME ?
I need to produce Oxalate decarboxylase enzyme as a recombinant enzyme. I choosed my source organisim named bacillus subtilis 168 designed my primers (with doubt) and I want to express in pET-SUMO vector. Are there anyone who tried this vector to produce protein for enzymatic reaction ? OR Can you please suggest me a vector system to produce protein for enzymatic reaction ?
I've never used the pET-SUMO expression vector before but I read on the Life Technologies that it produces high levels of soluble protein, so it might be OK.
In the past I tried to express an enzyme from a different Bacillus strain in E. coli and got very low levels of expression using a different type of pET vector. To solve this problem, I first tried expression in the RosettaBlue(DE3) strain that provides rare E. coli codons which helped somewhat (since codon usage differs between organisms). But it wasn't until I had the gene codon optimized for expression in E. coli that I got very high yields. However, this is expensive, so it might be better to first try it the traditional way. Good luck!Following
- Guillaume Pezeron added an answer:What do you use as a positive control when performing Tol2 experiment?
I've created a construct for Tol2 Gateway enhancer assay and am about to proceed with experiments. However, I do not have a positive control (nor negative). Can you suggest me what would be the best thing to use?
- Uriel Barboza Perez added an answer:Have there been any bioluminescent plants created?Have the biosynthetic pathways, leading to the production of luciferin and the genes responsible, been identified?
Sean! i saw this project when it was still a kickstarter iniciative.. It was supposed to be done on late 2014, but i haven't heard any news on it. There is a lot potential in using a plant synthetic biology approach and I hope they do achieve it ,or at least lay a foundation for someone else to do it.Following
- Kai Schönig added an answer:Did anyone used an inducible system that express rtTA ubiquitously in transgenic mice?
We are engaged in a metabolic project where we want to express our gene in a whole body and inducible fashion (or at least in important metabolic tissues as muscle, WAT, BAT and liver).
Do anyone know a model to express rtTA ubiquitously?
Hi, I would look at this line: http://jaxmice.jax.org/strain/016532.html
reference: Premsrirut PK; Dow LE; Kim SY; Camiolo M; Malone CD; Miething C; Scuoppo C; Zuber J; Dickins RA; Kogan SC; Shroyer KR; Sordella R; Hannon GJ; Lowe SW. 2011. A rapid and scalable system for studying gene function in mice using conditional RNA interference. Cell 145(1):145-58. [PubMed: 21458673] [MGI Ref ID J:171191]
However, in this paper the line is only shortly described. But as far as I know it is the best one for broad expression.
You can also have a look here:
- Jaya Gosala added an answer:What is the best way to amplify a 5kb+ gene with 75% GC content from genomic DNA?
I have been trying to amplify 10 different genes that vary in size from 4.5kb to 6.1kb in length from genomic DNA, but the genomes of these organisms are extremely GC-rich (73-75% GC). This holds true for the genes I wish to amplify as well. I have tried 4 different polymerases (Taq, Phire, Phusion, and Kapa) with multiple sets of primers. I have gotten the "best" results with Phusion with longer primers, but the Tms are still in the mid-70s for some, but there is a lot of background amplification. DMSO (from 3% to 15%) has helped some, and occasionally I see weak bands for these genes, but the results are not reproducible, and I have not been able to amplify off of these either. Additionally, I have been adding restriction sites to each primer, but the extra purification step for this with such small PCR yields is a concern as well. Any advice would be greatly appreciated!
You can amplify the gene into small fragments ( without restriction digestion design primers at specific length with over hangs) and do the overlapping PCR to join the fragments.Following
- Vegard Eldholm added an answer:Any advice on two-step PCR library generation with respect to Taq polymerase and 3' A-overhangs?
I am trying to sequence (Miseq) a custom amplicon from many samples using the Nextera indexes. For those of you not familiar, this involves a two step PCR protocol. The first round of PCR is carried out with your custom primers that have 5' overhangs complementary to the nextera indexes. The second round of PCR adds the indexes and adapters. The issue I am having is that my primer set contains inosine and so I can only get amplification using a Taq polymerase (Accuprime Taq in my case). This leaves my 1st PCR product with 3' A overhangs and I am not sure how this will effect the 2nd PCR step.
If I use a proofreading enzyme in the 2nd step, will this remove the 3' A's or do I need to include a specific step in my protocol to blunt my 1st round amplicons?
Could I accomplish blunting by using a proofreading polymerase and including a 72 degree incubation before the initial denaturing step in my 2nd round of PCR? Do I need to do a specific blunting step and then an additional PCR cleanup before round 2 of PCR?
I agree with Petre. The a overhangs should have no impact in the end. You can go about as planned. Once you do the second round of amplification, introducing the indexes, the new amplicons will not contain the 3' overhangs as as these are not included in the indexing primers.
- Amanda Solem added an answer:Why transformation pNL4,3 E-R- Luc+ plasmids cannot pick up the right clone all clone?
We recently transformation pNL4,3 E-R- Luc+ plasmid(about 16kb) with Trans-1 competent cells(our lab made it). We picked ten more clone to perform digestion identification but all clones are negative. At first we suspected that the plasmid is contaminated, so we take a gel purification but it still failed. What's the major concern in transformation this plasdmid? The competent cell line should be a important issue?
It might be helpful to use a strain designed for large plasmids like this: https://www.neb.com/products/c3019-neb-10-beta-competent-e-coli-high-efficiency.
Also, check out this previous question:
- Phil Geis added an answer:Is it possible to change the host range by transforming symbiosis genes of fungi to favorite host?
I mean truffle as fungi and some shrubs as host.
Not sure how one would know the design of "genes" so as to change the host range.Following
- Steve Sheardown added an answer:While adding IRES sequence between two different genes, how do I design the suitable IRES sequence?
I want to construct a bicistronic vector, which can co-express two different genes in Drosophila S2 cells. One way is to add IRES sequence between two genes, how to design the IRES sequence? Is it related to the genes I want to expressed? Thank you very much.
You can easily get suitable IRES containing plasmids from Addgene. I'd advise cloning it rather than attempting to PCR amplify as this can be difficult due to a very GC rich clamp at one end. IRES or IRES2? Doesn't make a lot of difference really, either way you will get a reduction in translation of the downstream cistron, typically about 70% the level of the upstream one. Personally I prefer 2A as it is very short and can even be incorporated onto the end of a primer to facilitate PCR-based cloning. The advantage of IRES is that you don't have to worry about reading frames (but do keep the ATG at the start of your downstream cistron) while 2A has to be in-frame. Expression is more stoichiometric with 2A, however there can be a low incidence of failure to cleave between the two peptides which effectively leaves you with a small amount of fusion protein. Usually that is not a problem but it's worth being aware of. Hope that helps.Following
- Jai Ghosh added an answer:Recombinant protein Expression in E.coli: Which gene's expression levels can be used to monitor the developed metabolic/cellular stress inside E.coli?
During recombinant protein expression (RPE) cellular/metabolic stress is developed inside E.coli, which then depresses the RPE over time.
Over the past few decades, several genes have been identified which can be used as a biomarker or probe to monitor this stress e.g. ppGpp, whose expression level increases with increased oxidative stress developed inside cell. Like this there are various stresses e.g. heat stress, osmotic stress etc. which are well correlated with the RPE.
I am trying to catalogue all those genes which can be used to monitor different type of stresses developed during RPE in E.coli.
Your valuable suggestions, ideas or Paper references will be well appreciated.
In E. coli infact this is a challenge for RPE gene. Note normally heat stress or osmotic stress do not give satisfactory results. You will get good results if you use oxidative stress.Following
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.