Genetic Engineering - Science method

Hi,

I'm transducing Ly1 and L363 cell lines using our standard protocol for retroviral transduction. The cells are successfully transduced as evidenced by GFP expression. However, after 4-5 days they start dying off and look really stressed. I'm suspecting polybrene since we've got a new batch. The cells look really weird, irregular and start forming clumps which they don't normally do in standard cell culture. I've tried using the same polybrene concentration (8ug/ml) in standard culture medium without the virus to check toxicity and it appears that it is decreased. Which concentrations do you normally use? Should I make a polybrene concentration curve to find the minimal nontoxic condition?

During Dolly's cloning, somatic cells were taken from one sheep, oocytes were taken from another, and injected into a third sheep.

Why was the somatic cells and the oocytes not taken, as well as the injection inside one sheep ?

Long story short, I need to degrade 30ug of RNA and i need to do it at 4C. I want to use only as much RNAse A as necessary.

So if performing the reaction at 4C, how long of incubation time and how much RNAse A would i need to degrade 30ug of RNA?

For example, would 1ug of RNAse A(20ug/ml conc.) for 15min at 4C be enough?

I have introduced 1 base mutation which cause 1 amino acid changed by PCR then transformed this plasmid to DH5-alpha. At this step, I checked desire transformants byspread on selective media (LB+Amp) and picked up some colony for DNA sequencing. After I got the right point mutation plasmid, I transformed it to yeast using auxotrophic marker (SD without Ura). I've got lots of yeast transformants, this come to my question, how can i make sure that my desire plasmid is transformed to the yeast cell. Another question is about the molecular knowledges that my yeast transformant contains its original gene and the mutate gene from plasmid, how can i determine that this mutate gene is affected to cell metalism since there is another original gene in the cell.

I want to know which institute's in India offer synthetic biology courses>?

Probiotics can be used as dietary supplements or drugs to treat diseases. The genetically modified probiotics will offer much more beneficial features than the wild type probiotics. Does FDA approve genetically modified probiotics? When do you think they will approve? What scientific questions do we need to resolve before FDA can approve? Can we use genetically modified probiotics as dietary supplements without FDA approval?

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What is the remarkable aspect of using phage promoters?

I have used wild type E. Coli BL21 DE3 (non-transformed cell) as control for my fluorescence experiment and measured the fluorescence value in a spectrofluorophotometer. As time increases, control fluorescence value also increased along with growth of the cell. I am curious to know how come the wild type E. Coli BL21 DE3 generates value in spectrofluorophotometer as it does not harbor any plasmid? Kindly help me to overcome the control fluorescence.

Genetic Engineering, Crisper

As SSR are most widely used markers in major cereals. They are highly reliable (i.e. reproducible), co-dominant in inheritance, relatively simple and cheap to use and generally highly polymorphic. What other factors can affect polymorphism in case of SSR markers?

There has been many occasions where people do not expect to be answered in terms of engineering, especially in biological sciences. However, it is more common to find the answer in terms of an engineering only in biological sciences. We have reached a point where it is impossible to separate, the engineering from the biological discipline.

The most common one is genetic engineering. How many more engineerings are there and can anyone explain the differences and what they are?

I would like to add the engineerings I can think of, but I am afraid I am not an expert on these areas, as I still need to give a definition to all of them. I will appreciate if anyone could complete the definitions, and make a list of them.

Is there anything else on these engineerings?

Any contribution is welcome.

In case the normal sequence of a gene which is existed in the gene bank contains a G, but the wild type of the gene contain an A instead, does this mean that the mutant allele becomes the wild type allele ? Can this occur ? If yes. then how much time this needs?

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I am creating CRISPRi tool for Lactococcus lactis. Do I need to add an extra Lactococcus Terminator sequence at the end of the sgRNA or is the S. pyogenes Terminator enough?

Question pertains to comparison of level of mRNA expression from single gene copy in the genome to single-copy-single-plasmid system.

Kindly also explain the reasons for the same.

Hello,

If I want to clone a gene of interest in a plasmid containing ORF, how should I do that, and which criteria I should follow?

Thanks,

Ramanjaneyulu

I intend to fuse my gene of interest (apoptin) with GFP using pCAMBIA1302 vector. I figured that I can do that by adding NcoI and SpeI restriction sites to 5' and 3' end of my GOI using PCR and later insert it into the plasmid.

However, as you can see in the image, 3 base pairs within SpeI restriction site do not overlap with the GFP gene and thus will be translated as an amino acid that is neither GFP's nor the GOI's. Will it disrupt my fusion protein structure/function? Thank you.

Can we transform any gfp tagged plasmid vector in to any E.coli strains with a objective to visualize E.coli bacteria as fluorescence molecule?. I need a valuable suggestions so Please help me. Thanks!

Could anyone give valuable inputs of this strain nomenclature F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), λ-, ΔpotD783::kan, rph-1, Δ(rhaD-rhaB)568, hsdR514?. Actually I need only pot D mutant bacteria having kanamycin resistant!. This strain is mentioned on CGSC Strain as JW1109-1on yale stock culture center.

Could you recommend a transposase containing plasmid with the SP6 promoter for making mRNA of a transposase enzyme to target the Tol2 sites for making transgenic zebrafish

Need help with my Genotyping. I cannot get WT bands to show up on gel.

i need to transform this strain, kindly suggest me some technique with previously published information about transformation efficiency...

I am trying to express and isolate a protein in E. coli. The protein is supposed to have high prevalence of non-polar residues. Would that create problems in isolating the protein using a His tag column? If yes, are there other ways we can isolate proteins which are rich in non-polar residues?

Hello Everyone, Please share your experience. Thank you.

Golden Rice is a GMO rice plant that was modified to produce beta-carotene (provitamin A) in its grain. It was invented by the German scientists Prof. I. Potrykus (ETH) and Prof. P. Beyer (Univ Freiburg) in 2000. In December 2019, the Philippines joined Australia, Canada, New Zealand, and the USA that accepted the use of this genetically-engineered crop. Do you think Golden Rice is a miracle technology that will help solve hunger and malnutrition, especially in developing countries?

A biosafety level is the level of the biocontainment precautions required to isolate dangerous biological agents in an enclosed facility. The levels of containment range from the lowest biosafety level 1 to the highest at level 4. In the United States, the Centers for Disease Control and Prevention (CDC) have specified these levels. In the European Union, the same biosafety levels are defined in a directive. Sabanci University is following the same directive in accordance with Turkish biological safety regulation.

Genome sequencing helps find vital information, for example the strain type, virulence, location of origin and differences between strains transmitted within the country and in other countries

FDA has issued guidance to provide recommendations to health care providers and investigators on the administration and study of investigational convalescent plasma collected from individuals who have recovered from COVID-19 (COVID-19 convalescent plasma) during the public health emergency.

The guidance provides recommendations on the following:

Because COVID-19 convalescent plasma has not yet been approved for use by FDA, it is regulated as an investigational product.  A health care provider must participate in one of the pathways described below.  FDA does not collect COVID-19 convalescent plasma or provide COVID-19 convalescent plasma.  Health care providers or acute care facilities should instead obtain COVID-19 convalescent plasma from an FDA-registered blood establishment.

Suppose i have a DNA sequence and i want to find transcription strat site, CDS, poly A signal etc., which software will be useful to find this out?

Biological techniques are methods or procedures that are used to study living things. They include experimental and computational methods, approaches, protocols and tools for biological research.

CRISPR (/ˈkrɪspər/) (clustered regularly interspaced short palindromic repeats) is a family of DNA sequences found in the genomes of prokaryotic organisms such as bacteria and archaea. These sequences are derived from DNA fragments of bacteriophages that had previously infected the prokaryote. They are used to detect and destroy DNA from similar bacteriophages during subsequent infections. Hence these sequences play a key role in the antiviral (i.e. anti-phage) defense system of prokaryotes .

CRISPR gene editing is a genetic engineering technique in molecular biology by which the genomes of living organisms may be modified. It is based on a simplified version of the bacterial CRISPR-Cas9 antiviral defense system. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added in vivo.

As using A.Tumefacinies for inserting gene X and A.Rhizogenes for gene Y (in my case Y is for CRISPR cas9).

The SARS-CoV-2 genome was rapidly sequenced by Chinese researchers. It is an RNA molecule of about 30,000 bases containing 15 genes, including the S gene which codes for a protein located on the surface of the viral envelope (for comparison, our genome is in the form of a double helix of DNA about 3 billion bases in size and contains about 30,000 genes).

Comparative genomic analyses have shown that SARS-CoV-2 belongs to the group of Betacoronaviruses and that it is very close to SARS-CoV, responsible for an epidemic of acute pneumonia which appeared in November 2002 in the Chinese province of Guangdong and then spread to 29 countries in 2003.

A total of 8,098 cases were recorded, including 774 deaths. It is known that bats of the genus Rhinolophus (potentially several cave species) were the reservoir of this virus and that a small carnivore, the palm civet (Paguma larvata), may have served as an intermediate host between bats and the first human cases.

Since then, many Betacoronaviruses have been discovered, mainly in bats, but also in humans. For example, RaTG13, isolated from a bat of the species Rhinolophus affinis collected in China's Yunan Province, has recently been described as very similar to SARS-CoV-2, with genome sequences identical to 96 percent.

These results indicate that bats, and in particular species of the genus Rhinolophus, constitute the reservoir of the SARS-CoV and SARS-CoV-2 viruses.

Novel Coronavirus thought to have transferred to Human from the seafood market in Wuhan, China become a one of the most dangerous viruses in the subfamily Orthocoronavirinae. According to the literature, the genome size of RNA of this viruses are greater than 20 kilobases.

Genetic engineers has committed to change the genes of some organisms to create new features of them, and this can be applied for the Coronavirus as well.

I would like to discuss this matter with Genetic Engineers, Biologist and Scientists.

Previously, I had read a mechanism about conditionally-skip transcription from a cloned cassette in an expression vector. But i can't find that paper. It was containing a cis-regulatory element as I remember.

Any one knows that mechanism?

Thanks in advance

Rice is the seed of the grass species Oryza sativa (Asian rice) or Oryza glaberrima (African rice). As a cereal grain, it is the most widely consumed staple food for a large part of the world's human population, especially in Asia. It is the agricultural commodity with the third-highest worldwide production (rice, 741.5 million tonnes in 2014), after sugarcane (1.9 billion tonnes) and maize (1.0 billion tonnes).

Hello

I am working on CD19-directed CAR T-cells and using CD19 expressing cell lines for cytokine and killing assays. I want to include different cell lines that have different levels of antigen expression i.e. high, intermediate, low and none. Most of cell lines I have stained were highly positive for example Granta-519, Nalm-6, SUDHL-4, RAJI and T2. I looked online for Daudi but it seems to be also high. I have H929 as a negative.

Can anyone suggest cells that express intermediate and low levels of CD19 ?

Thanks

Abdulrahman

Recently there have been several studies that show, using a truncated sgRNA with a wild type CAS9 can deter the nuclease activity and can potentially be used for epigenetic regulation.

Kiani et al.(2015) claimed that 'that the lack of DNA cleavage with 16-nt gRNA was due not to a lack of DNA binding but to an inability of Cas9 to cleave the target substrate after binding.'

I wish to know how and why does the size of sgRNA affects the nuclease activity.

Hello All,

I am working on trying to increase the rates of successful integration of a transgene in catfish using CRISPR HDR knock-in. Right now we are using microinjection on eggs shortly after fertilization and have had great success for knock-out and have some success with HDR knock-in. Now we wanted to try to increase the integration rates for CRISPR HDR knock-in using SCR7 pyrazine and RS-1.

The question I have is has anyone tried microinjection of SCR7 pyrazine and RS-1 and if so what concentrations did you use?

The reason we want to use microinjection instead of incubation is that we have to incubate our eggs in a modified Holtfreter's solution (contains NaCl, NaHCO3, KCl, MgSO4, CaCl2, and Doxacycline) in order to prevent fungal growth and strengthen the eggs and I was not certain what effect this would have on SCR7 pyrazine and RS-1 if we add it to the solution.

Any suggestions are greatly appreciated.

Best Regards,

I am looking for a way to genetically engineer E. coli to display a custom peptide sequence on its outer membrane. I have read about the Lpp-OmpA surface display system (among others), and it seems like a good choice for me but I have not found any commercially available vectors/plasmids/strains or any detailed protocols online on how to create this model. I would be really grateful if someone could provide me some useful advice or resources on this matter.

we need Image Lab™ Software #1709690 for bio rad to visualize chemiluminescence, the original software actually got corrupted and getting from company is quite costly any other way to get it?

Of course, one can just suggest that, I should club the 3 genes together under a single promoter. But, in the particular genetic circuit that I am making, I need them to be independent of each other. Also, since I want a single input controlling them, I want to have the same promoter for all of them. So do I need to induce with 3x the normal inducer concentration? Also, what kind of other problems can I face here? Thanks in advance.

Hi everyone,

A gene is present in an "organism A" and its sequence can be found online on NCBI. An orthologue of the same gene is present in "organism B" but can't be found on the NCBI. Can I use the gene sequence of that "organism A" to design my primers and amplify the orthologue of the gene present in "organism B"? Both organisms belong to fungi but are from different classes.

Hello everyone,

I have two questions, please shed some light on this. I am new to in vivo studies and some may find my questions are trivial. TIA.

My first question is,

I wanted to develop the MCF7 mouse xenografts. I did a search and came to know that the injection of estradiol is recommended to develop tumours prior to injecting the MCF7 cells. Also, I have seen some papers without estradiol. Can you tell me the role of estradiol in tumour formation? Can the mice form tumour without estradiol?

Lastly,

Once the tumour reaches a definite volume, I wanted to inject my compound of interest. Here is the question, which is a simple way to inject? I have found the following ways related to my studies.

1. Trochar

2. Microosmotic pump

3. Intraperitoneal

4. Oral gavage

Please recommend me a simple way to introduce my compound to the mice. If you have a better option also welcome.

Many thanks in advance,

Arun.

I know this is a very broad question, but in your experience how sensitive is qPCR?

It is reported that qPCR can detect even 1000 CFU/ml or less of bacterial species in human samples, and brochures of qPCR manufacturers show that it is quite sensitive.

But, generally speaking (regardless of the type of master mix you are using), have you ever had sensitivity problems with qPCR and what were the lower limits of detection for you?

Cre recombination efficiency often impacts activation of Cre/LoxP dependent transgenes. We have recently encountered problems with recombination in certain cell types in vivo, especially with inducible recombinase (CreERT2). The recombination rates range from very high (>90%) to barely detectable. Increasing Cre concentration/action time by prolonging TAM induction regimens seems to improve it, albeit very modestly. Our transgene is in the Rosa26 locus, which many regard as a safe harbor. Interestingly, the same Cre recombinases (Cx43-CreERT2, Calb2-CreERT2, were amongst the problematic ones) seem to perform much better in other loci. We are considering two possibilities:

- methylation of the gene - this may effect recombination (e.g., if LoxP sites are methylated), but may be also happening independently

- closed chromatin leading to reduced Cre access to the locus and/or reduced overall expression of the gene

In any case, I am toying with the idea of using an alternative STOP cassette, flanked by 3 LoxP sites on each side, to increase the efficacy of recombination. It may not help if the issue is not cre related, but I don't see any risk with this approach. A slightly higher chance for random recombination seems a small price to pay if using such LoxP triplets (all facing in the same direction) would help. I would appreciate feedback from someone more experienced, about pros and cons of this solution.

On a related note: I am curious about the relative efficiencies of Cre, Flp and PhiC31 on different target sequences (e.g., LoxP vs Lox2272, AttP-CT/AttB-CT vs AttP-TC/AttB-TC, etc.). I would especially interested in strategies to optimize Cre/LoxP and Flp/FRT system, but I am also considering PhiC31 for the same purpose (especially that the latter works in unidirectional way - I see this and an advantage - and this paper shows that PhiC31 may be superior to Flp). I am aware of the one report discussing PhiC31 causing (potential) DNA damage, but I would love to hear more opinions. Regrettably, the literature mentions the above mentioned issues are very sparse; I failed to find any relevant recent papers. I would be delighted to hear expert opinions of fellow researchers.

Hi All,

I am trying to generate a bacterial operon construct. I have designed four gBlocks (each of which is ~1.7 kb) and synthesized them from IDT. I am using pGex-6p-1 vector to assemble these gBlocks using Gibson assembly. I prepared my vector by PCR (primer designed by NEB builder software). After PCR amplification, I checked 5 ul of my PCR product in the gel and found a nice clean single band and the size of this band matches to the expected size (for details see the attached PPT slide). After that, I purified the PCR product using Promega PCR and gel purification kit. I eluted in 30 ul of nulcease free H2O and then measured the concentration of PCR product using NanoDrop and the concentration was 296 ng/ul. Then, I digested 8 ul (296 ng/ul) of purified PCR product with DpnI (1 ul) in a 10 ul of total reaction volume (and incubated at 37oC for 30 mins) as suggested by NEB Gibson manual and heat inactivate the DpnI at 80oC for 20 mins. After that, I set up a Gibson assembly following the instruction of NEB Gibson manual. I incubated the Gibson reaction at 50oC for 1 hr in a PCR machine and then transformed 2 ul of assembly reaction in 50 ul of NEB 10-beta cell (High efficiency) following the transformation protocol for NEB beta cell as specified by NEB. In the following day of transformation, I found many colonies (see attached PPT for picture). I took five well separated colonies and then grew a 5 ml overnight culture. After that, I isolated plasmid DNA using Promega plasmid purification kit and digested them with SalI-HF enzyme which should give three bands if there is a assembled product. Otherwise, they should produce linear vector as pGEX-6p-1 has an internal SalI site. I then checked 60 colonies by colony PCR and it seemed that all of the colonies I checked by colony PCR contains the intact pGEX-6p-1 ( I did not include this gel image in the PPT slide). What things might be wrong with my cloning? Specifically, I would like now

i) Why I am getting intact pGex-6p-1 whereas I generated the linearized the pGex-6p-1 by PCR and digested the PCR product with DpnI? Is digestion not working??

ii) What might be the good way to assemble these four gBlocks into pGex-6p-1 vector?

iii) How can I reduce the vector only background?

iv) I would also highly appreciate any suggestion on my strategy that I am using to generate this operon construct.

For details description of my cloning strategy, please see the attached PPT slides.

Thank you all,

Hassan

I am planning an experiment where I will integrate an operon construct in the gfc locus E. coli using CRISPR-cas9 mediated homologous recombination. To insert my operon construct in the gfc locus, I have included a portion E. coli endogenous gfc sequence on either side of my operon construct so that homologous recombination become induced upon induction of Cas-9 mediated double-stranded break at the gfc locus. However, my worry is that presence of gfc locus in my operon construct might create problem during cloning as the gfc sequence should also be present in the E. coli strain commonly used for cloning. It may be that portion of gfc in my operon construct and one that is present in E. coli cloning strain might undergo recombination and make this construct unstable and difficult to made. Initially, I planned to synthesize them from a company, however, later I feel the company might face the same problem. I have only 5 months to complete my project and thus I am looking for an efficient and quick way of making my operon construct. Any suggestion on how can I design a better strategy to make this construct will be highly appreciated. For easy understanding, I have uploaded my construct design here. Thanks.

There are many ideas to create genetically modified microalgae for higher productivity,

Why are the reports on this so little?

I think now it is more possible with CRISPR technology.

Any idea of what are the barriers?

I have tried searching everywhere but did not find the source. SspI cuts at AAT^ATT

I am attempting to express GFP in S. cerevisiae using the GAL1 promoter. I always grow an uninduced (in dextrose based SD medium) culture alongside my induced (in galactose based SD medium) culture. I see approximately the same very low level of fluoresence in both cultures, and also a band at 27kD on a PAGE gel. There is no overexpression of the GFP happening, the fluorescence is very weak by eye, and the cultures are not green in visible light, like E. coli cultures overexpressing GFP. On the PAGE gel the amount of protein appears to be the same in both the uninduced and induced samples. I need to overexpress the protein, this leaky level of expression isn't acceptable. How do I go about fixing it? I am currently growing up a culture to do a plasmid prep to send for sequencing to make sure the promoter region of the plasmid is intact.

I'm planing two use two different genes in E.coli cells, So I would to know which is better? To get both CDS under different promoters in one plasmid or to insert each gene in a separate compatible plasmid?

Also, one of these gene is RFP to get colored cells. So, I'm planing to insert it at first and use the RFP cells to prepare a competent cells, then these competent cells can be used for transformation of any further gene. Is there any problem or suggestion with this strategy?

Thanks in advance :)

When we have a positive regulation the control is tight, so we have low background expression under non-induced conditions.

Negative regulation can not be fully controlled, but as far as I understand it is more popular than positive regulation.

Why?

Are there any other important features of these two types of regulations?

Thank you in advance!

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 ?

Dear all,

I am currently trying to run the MASURCA assembler with my Illumina paired-end reads. The first steps work fine but when it comes to the error correction with Quorum the program reports the following error:

quorum_error_correct_reads -q $((MIN_Q_CHAR + 40)) --contaminant=/gpfs0/global/local/masurca/3.1.3-1/bin/../share/adapter.jf -m 1 -s 1 -g 1 -a 3 -t 16 -w 10 -e 3 quorum_mer_db.jf pe.renamed.fastq --no-discard -o pe.cor --verbose > quorum.err 2>&1

+ fail Error correction of PE reads failed. Check pe.cor.log.

However, there is no pe.cor.log produced. A Google research on that issue was also not very helpful. Thus, I would very much appreciate any suggestions/comments/reports.

I was also wondering where to specify the Illumina version used when running MASURCA?! Could it be that the Quorum error correction assumes the wrong Illumina version, i.e. uses the wrong quality code? Thanks a lot.

Best,

Belinda

Qiagen launched their NGS platform GeneReader in Nov 2015, i am curious if anyone can allready comment on it.

I want to clone gene fragment into the vector pT1NX in order to intracellularly express it. According to the instructions provided by BCCM/genecorner plasmid collection, for intracellular expression the fragment can be cloned into the unique BglII site. However, in this way of cloning (using one restriction enzyme) there is the risk of the fragment being cloned in a wrong direction (in about 50% of clones). Can anyone experienced with this vector tell me if I can clone my gene fragment (with a functional Ribosome Binding Site or RBS) into the vector using two restriction enzymes BglII and SpeI (which removes usp45 signal sequence and SpaX fragments from the vector) instead of cloning it into the unique BglII site in order to get a intracellular expression and circumvent the risk of cloning in the wrong direction?

Enclosed I have attached the vector map

So I always have this weird results during my gene cloning experiments. I perform restriction enzyme digestion (double digest) for my plasmid vector (7.0 kb) and insert gene (1.5 kb). I perform ligation using T4 DNA ligase, incubate for 16 h at 16 C, then E. coli DH5a transformation with proper controls.

After picking colonies, plasmid miniprep, I get pDNA transformants with (circular) size smaller than the control (so many of them). I always get this kind of results even in the past, I can't get proper transformants and one of the problem is this and the other is low transformation efficiency. I am not sure what is the problem? Can anyone help me?

To me it's a host problem, when I switch to E. coli HST08 from DH5a, I get better results and higher rate of successful transformants. But some colleagues said my DNA concentration is too low, although I've been using 150-200 ng of DNA vector and the 5:1, 10:1 or more insert:vector molar ratio even in the past. Any one with the same problem?

I am in an academic research project, where we want to introduce a gene that codes for obtaining an enzyme that degrades phenols in the organism P. Infestans, so we want to introduce a construct together with the enzyme of interest in the organism, and this It leads me to ask myself the following question: We want to introduce the gene near the ribosomal site since it is a very conserved region and therefore this would prevent the gene from being maintained in the organism, so could this be possible? Or is it better? place the construct in another part of the body's genome?

Greetings.

I am planning to express a recombinant protein in Ecoli and want it secreted extracellular (into the media). I have been reading that Gaussia's sec signal would work in prokaryotes as well. I wonder if the secretion signal is cleaved or not.

Also, do we know if it follows sec or tat pathway?

Any help would be appreciated.

Thank you

Hello,

I would like to know why some vectors contain this AAV2 ITR sequence, I want to remove that sequence together with others of my vector to make the stable transfection with lipofectamine more efficient. My question is, if I remove this sequence, would it affect the stable transfection of my fibroblasts?

What is the function of this AAV2 ITR sequence in vectors?

Thank you very much in advance!

Is it possible to use gamma irradiation to induce polyploidy on plants?

The question is part of a project in the intersection of science, art and design that examines genetic identity. The aim is to create a "genetic ghost" by altering the genetic information of a DNA sample at loci that are necessary for DNA fingerprinting.

[Image by Dr. Thomas Splettstößer www.scistyle.com]

Genetic engineering of food crops can produce a whole range of positive properties from making the food more nourishing, less attractive to parasites, longer shelf life and more resistant to extremes of climate.

If climate change affects our ability to grow crops for the hugely expanding world population are we finally going to have to abandon our often irrational fears of GE foodstuffs and embrace genetic modification as a necessary adaptation to climate change?

Hi all,

I have a validated sgRNA to KO our gene of interest in the adult mice brain And I was thinking of using Lenticrispr V2 with mcherry and to inject it ICV, has anyone had any experience with such a project? I couldnt find any relevant articles.

Do you think it would be better to just inject a virus with only the sgRNA into the brain of a cas9 KI mouse?

Thanks in advance :)

Which of the plasmid copy number detection protocol can help? Protocols on net ? Can anyone specify any ideal protocol? Please send some recent reviews or papers even in animal system if you have..A good statistical explanation is what I am also looking for..

Hello,

I recently bought a genetically modified e.coli (pje202/pVIB plasmid), which is bioluminescent. I don't want to grow the e.coli on antibiotic agar forever, because the antibiotic selection has many limitations. The plasmid produces b-lactamase, so it is resistant to f.e. ampicillin, carbenicillin etc.

If I don't grow it on lb-amp, it will lose its plasmids. I wondered, whether I can slow down the plasmid loss, by growing the bacteria on drug-less lb agar, then isolate the brightest colony, regrow it on lb-amp and then again grow it on pure lb and isolate again. I thought, this way I may isolate the mutants that are less likely to lose plasmids, because bacteria containing the plasmid will glow only.

Thanks very much,

regards,

André

I recently bought pVIB plasmid modified e.coli (BL21 strain) and now I want to grow them on LB agar containing ampicillin for selective pressure. I wondered, whether there is a chance due to spontanous mutations that a non plasmid containing e.coli might survive on the amp agar?? Could it ruin my culture?

What can I do to prevent it?

I recently bought a pre-modified e.coli bacterium containing pVIB plasmid.

The plasmid is resistant to ampicillin, but I wonder if there is a possibility to grow this bacterium without selective pressure. Maybe integrating the plasmid into the bacterial dna to prevent plasmid loss any ideas?

Hello,

I want to take a timeout from my other project and now I am into genetically engineered bacteria. I want to use the pje202/pVIB plasmid containing e. coli. This bacterium has to grow on ampicilline or it will eventually lose the plasmid. I wanted to ask, whether there is another possibility to prevent plasmid loss other than using antimicrobial drugs?

Thanks very much,

regards,

André Leonhardt

Betalains and anthocyanins are said to be mutually exclusive in a plant species (Stafford HA, 1994, Plant Sci. 101:91-98). However, Harris et al. (2012, BMC Plant Bio 12:34) reported that betalains can be induced in anthocyanin-producing plants through genetic engineering and substrate feeding, possibly because of a background enzyme. 

1. Alternatively, can betalains be induced in plants that normally produce anthocyanins in an in vitro system using plant growth regulators?

2. Would spectrophotometric analysis be sufficient to detect the presence of both pigments in any given sample?

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.