Science method

Genetic Engineering - Science method

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.
Questions related to Genetic Engineering
  • asked a question related to Genetic Engineering
Question
6 answers
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?
Relevant answer
Answer
My problem was solved by switching to another HEK cell line
  • asked a question related to Genetic Engineering
Question
1 answer
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 ?
Relevant answer
Answer
The driving force for producing Dolly was not to produce genetically identical animals. Rather researchers wanted to combine cloning techniques with other methods in order to efficiently change animals genetically, much quicker than traditional animal breeding methods that take decades to make changes in populations of species. So, somatic cell nuclear transplantation became an essential part of the process.
Dolly was produced by somatic cell nuclear transfer process. In this process, researchers removed the genetic material from an egg and replaced it with the nucleus of some other body cell. The resulting egg became a factory to produce an embryo that developed into an offspring. So, Dolly was identical to the Finn Dorset ewe that donated the mammary cell only in terms of nuclear genetic material, but clearly different with respect to the micro- and macro-environmental factors to which it was exposed, like the conditions depending on the uterus containing the embryo.
Therefore, Dolly was produced from three different sheep of two different breeds. Her “genetic mother”, the sheep from whom she was cloned in the common understanding of the term, was a Finn-Dorset. Dolly’s “second mother”, a sheep that was a Scottish Blackface, which donated an unfertilized egg cell, and Dolly was born to her Scottish Blackface surrogate mother which was her “third mother", with the use of standard in vitro fertilization techniques.
The ultimate goal is to refine techniques and combine them with other methods to turbocharge traditional animal breeding methods as well as gain insights into aging and disease.
Best.
  • asked a question related to Genetic Engineering
Question
3 answers
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?
Relevant answer
Answer
I agree with Kyle Skalenko that experimentation is needed but first contact the company to sort out whether they work in enzyme units or Kunitz units to define the activity of your rnaseA, Then assume that the activity drops by a factor of 2 for every 10c drop in temperature to get an approximation of how much enzyme or increased time will be needed
  • asked a question related to Genetic Engineering
Question
5 answers
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.
Relevant answer
Answer
Hi,
I will recommend you use a yeast mutant for the gene you mutated. That way, you can easily select your transformants and can determine their cellular consequences of your mutation.
  • asked a question related to Genetic Engineering
Question
4 answers
I want to know which institute's in India offer synthetic biology courses>?
Relevant answer
Answer
  • asked a question related to Genetic Engineering
Question
5 answers
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?
Relevant answer
Answer
The GM probiotics, like other GMO, have both their pros and cons depending on the method (s) used to make them.
  • asked a question related to Genetic Engineering
Question
16 answers
We are trying to build up a "Quick Step Guide for Young Scientists Playlist" to assist young researchers in their journey of scientific writing and publishing. Playlist link: https://www.youtube.com/playlist?list=PLwyWKnsS4EkgcQ1Wnkx7FIxUsLsLUp0gb
We hope it will help many of you.
Share it with your friends and colleagues. All the very best!! Channel link: https://bit.ly/Subscribe_Learn_SciTech
Relevant answer
Answer
Also these are good:
1- Writing Research Papers. Student's Book: From Essay to Research Paper
Dorothy E. Zemach, Daniel Broudy, Chris Valvona - 2013 - 128 pages
2- English for Writing Research Papers (English for Academic Research)
Part of: English for Academic Research (11 Books) | by Adrian Wallwork | Mar 3, 2016
  • asked a question related to Genetic Engineering
Question
2 answers
What is the remarkable aspect of using phage promoters?
Relevant answer
Answer
I think the identification of regulatory elements, primarily promoters, which are specific DNA regions responsible for transcription initiation. Muskan Gupta
  • asked a question related to Genetic Engineering
Question
8 answers
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.
Relevant answer
Answer
This phenomenon has been nicely quantified here https://hal.archives-ouvertes.fr/hal-01628395/document
  • asked a question related to Genetic Engineering
Question
4 answers
Genetic Engineering, Crisper
Relevant answer
Answer
The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) method, I think it is ethical for therapy and acquired immunity.
  • asked a question related to Genetic Engineering
Question
11 answers
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?
Relevant answer
Answer
The polymorphism at DNA level depends on several factors of which genetic divergence of the parents or the genotypes under study is one of the most important one. Secondly, as very rightly mentioned by Ricardo Julian Licea-Moreno sir, it requires detailed information about the genotypes and the markers you are using because it is possible that many of those markers may belong to the same region of a specific chromosome.
  • asked a question related to Genetic Engineering
Question
24 answers
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.
Relevant answer
Answer
For Much Detail......it may helpful for you
  • asked a question related to Genetic Engineering
Question
8 answers
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?
Relevant answer
Answer
Kindly check the attached file
  • asked a question related to Genetic Engineering
Question
7 answers
.
Relevant answer
Answer
Recombinant Gene
  • asked a question related to Genetic Engineering
Question
4 answers
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?
Relevant answer
The answer to the question is in the article below.
  • asked a question related to Genetic Engineering
Question
6 answers
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.
Relevant answer
Answer
The short answer is "generally, yes".
The longer answer is to point out that copy number is only a single variable in a process controlled by many, many factors.
A single copy genomic gene with a strong promoter will give you far, far more expression than a plasmid gene with a weak promoter, even if you have multiple copies of the plasmid. A plasmid gene with a strong promoter that requires factors not present in the host cell will give almost no expression, even if you have multiple copies.
A single copy genomic gene might give you more or less expression than an ostensibly identical plasmid gene depending on whether the genomic copy lies in a more or less active region of the genome.
A single copy of a plasmid might express wildly differently between individual cells because of stochastic effects.
Biology is messy.
What is the specific question you are hoping to answer, here?
  • asked a question related to Genetic Engineering
Question
2 answers
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
Relevant answer
Answer
Cloning DNA fragment essentially involves this steps:
  1. isolation of the DNA of interest (or target DNA),
  2. ligation,
  3. transfection (or transformation),
  4. screening/selection procedure.
DNA fragment to be cloned must be isolated, you do with PCR or restriction enzymes isolated from bacteria, which are able to recognize and cut specific sequences creating "Sticky" or "Blunt" DNA ends.
The plasmid is digested with restriction enzymes, opening up the vector to allow insertion of the target DNA. If the isolated DNA of interest and the plasmid or vector are digested with the same restriction enzyme, their sticky ends will be complementary. The two DNAs are then incubated with DNA ligase, an enzyme that can attach together strands of DNA with double strand breaks.
Following ligation, the recombinant DNA is placed into a host cell like E. Coli, in a process called transfection or transformation with high concentration of calcium or heat shocks.
Finally, the transfected cells are then cultured but some may not contain a plasmid with the target DNA because the transfection process is not usually 100% successful and the appropriate cultures must be selected with markers usually for antibiotic resistance. When the treated cells are plated on a petri dish of nutrient agar containing the antibiotic, only the rare transformed cells containing the antibiotic-resistance gene on the plasmid vector will survive.
Further analysis of the resulting colonies is required to confirm that cloning was successful. This may be accomplished by means of a process PCR or restriction fragment analysis, both of which need to be followed by gel electrophoresis and/or DNA sequencing. DNA sequence analysis, PCR, or restriction fragment analysis will all determine if the plasmid/vector contains the insert. Restriction fragment analysis is digestion of isolated plasmid/vector DNA with restriction enzymes. If the isolated DNA contains the target DNA, that fragment will be excised by the restriction enzyme digestion. Gel electrophoresis will separate DNA molecules based on size and charge.
Some suggestions:
  1. Get a protocol from another investigator in the lab so you have skills and resources at your fingertips.
  2. Many laboratory manuals are commercially available with simple and clear protocols provided for many fields, but you will need to refine the protocol yourself.
  3. Methods section in published articles, last reliable place to find the protocol.
I hope I was helpful Golla Ramanjaneyulu
  • asked a question related to Genetic Engineering
Question
3 answers
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.
Relevant answer
Answer
I think you should be fine with this. Many times it is wise to put a linker region between your desired protein and the desired tag (here gfp). This helps to ensure proper folding and hopefully functionality between the protein of interest and the tag. Your linker amino acid here will not produce a stop codon, so the gfp should be transcribed, but you should always check to ensure your desired protein remains functional when adding a tag on the end of a protein. Just ensure that you do not include the stop codon in the PCR primers for your protein of interest, otherwise the gfp will obviously not be transcribed.
  • asked a question related to Genetic Engineering
Question
10 answers
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!
Relevant answer
Answer
As mentioned by Hanna Alalam it may depend a bit on the strain but in theory you should be able to transform nearly any E. coli strain. However whether you get good expression of the GFP will depend upon the construct itself, is there a suitable promoter and appropriate translation signals for GFP expression in E. coli.
  • asked a question related to Genetic Engineering
Question
6 answers
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.
Relevant answer
Answer
Hello, I recommend to consult Berlyn MK (1998) Linkage map of Escherichia coli K-12, edition 10: the traditional map. Microbiol Mol Biol Rev 62(3): 814-984 (see attachment). The Instructions to Authors of the Journal of Bacteriology as a great resource as well. They explain genetic nomenclature for bacteria.
  • asked a question related to Genetic Engineering
Question
2 answers
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
  • asked a question related to Genetic Engineering
Question
5 answers
Need help with my Genotyping. I cannot get WT bands to show up on gel.
Relevant answer
Answer
May I check what does WT and KO refers to ?
  • asked a question related to Genetic Engineering
Question
2 answers
i need to transform this strain, kindly suggest me some technique with previously published information about transformation efficiency...
  • asked a question related to Genetic Engineering
Question
3 answers
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?
Relevant answer
Answer
High prevalence of non-polar residue in protein (his-tagged) is not going to affect its purification by affinity chromatography. Membrane proteins have a high percentage of non-polar amino acids. You can purify the protein under non-denaturing conditions & run on an SDS-PAGE to check the presence of desired protein. If your protein is a membrane one, do not boil your sample before SDS-PAGE as it can form aggregates. Heat it to 70c for 15mins.
If the protein is not soluble, then you have to purify the protein under denaturing conditions & refold it.
  • asked a question related to Genetic Engineering
Question
5 answers
Hello Everyone, Please share your experience. Thank you.
Relevant answer
Answer
Vaibhav Sharma I used both Norgen for Urine and miRNeasy Serum/Plasma Kit from Qiagen for plasma and saliva. Concentrate and purity were quite fine in our samples.
  • asked a question related to Genetic Engineering
Question
12 answers
  • Molecular biology is the study of the molecular underpinnings of the processes of replication, transcription, translation, and cell function.
  • Biochemistry is the study of the chemical substances and vital processes occurring in living organisms. Biochemists focus heavily on the role, function, and structure of biomolecules such as proteins, lipids, carbohydrates and nucleic acids.
  • Genetics is the study of how genetic differences affect organisms. Genetics attempts to predict how mutations, individual genes and genetic interactions can affect the expression of a phenotype.
Relevant answer
Answer
For detailed study:
1) Molecular biology by ROBERT F WEAVER
2) Molecular Biology of the Cell by Bruce Albert et al.
3) Karp's Cell and Molecular Biology by Gerald Karp, Janet Iwasa, Wallace Marshall
4) Cell and Molecular Biology Concepts and Experiments by Gerald Karp
5) Molecular Cell Biology by Harvey Lodish, Arnold Berk, Chris A. Kaiser, Monty Krieger, Anthony Bretscher, Hidde Ploegh, Angelika Amon, Kelsey C. Martin
For outlines ( simplified book):
1) Molecular and Cell Biology For Dummies by René Fester Kratz
2) Schaum's Easy Outline Molecular and Cell Biology by William Stansfield, Raul Cano, Jaime Colome
3) Study Guide to accompany Cell and Molecular Biology: Concepts and Experiments, by Gerald Karp, Nancy L. Pruitt
  • asked a question related to Genetic Engineering
Question
55 answers
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?
Relevant answer
Answer
While it is absurd that it took so many years to be approved, by itself Golden Rice is not a universal panacea to feeding the world. It may be unpopular to say but no matter how much we improve crop yields and decrease food wastage, the only way to continue feeding the world in the future is to recognize that we live on a planet whose resources are finite and therefore limit the size of the global population.
  • asked a question related to Genetic Engineering
Question
3 answers
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.
  • asked a question related to Genetic Engineering
Question
9 answers
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
Relevant answer
Answer
You can find a centralized database of genomes on https://www.gisaid.org/ . To access them, you have to register and it can take some time to actually obtain the info. Nevertheless, you can see the authors of the publications and contact them directly.
  • asked a question related to Genetic Engineering
Question
14 answers
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:
  • pathways for use of investigational COVID-19 convalescent plasma
  • patient eligibility
  • collection of COVID-19 convalescent plasma, including donor eligibility and donor qualifications
  • labeling, and
  • record keeping
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.
  • asked a question related to Genetic Engineering
Question
11 answers
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?
Relevant answer
Answer
#GlimmerHMM is a new gene finder based on a Generalized Hidden Markov Model (GHMM). It actually helps you to annotate the draft genome by running a few simple commands.
  • asked a question related to Genetic Engineering
Question
34 answers
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.
Relevant answer
Answer
  • asked a question related to Genetic Engineering
Question
10 answers
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.
Relevant answer
Answer
Hi,
I'd add this one for the double nicking
Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity.
Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S, Trevino AE, Scott DA, Inoue A, Matoba S, Zhang Y, Zhang F.
Cell. 2013 Sep 12;154(6):1380-9. doi: 10.1016/j.cell.2013.08.021. Epub 2013 Aug 29. Erratum in: Cell. 2013 Oct 10;155(2):479-80.
PMID: 23992846
And if you want the history of the critical steps late 80's to the development of the bioengineering tool:
The Heroes of CRISPR.
Lander ES.
Cell. 2016 Jan 14;164(1-2):18-28. doi: 10.1016/j.cell.2015.12.041. Review.
PMID: 26771483
Enjoy
  • asked a question related to Genetic Engineering
Question
36 answers
As using A.Tumefacinies for inserting gene X and A.Rhizogenes for gene Y (in my case Y is for CRISPR cas9).
Relevant answer
Answer
Abdelrahman Ahmed Khalifa The reason I was asking you about root-specific promoters is that you can also do this way:
(1) transformation 1: use A.Tumefacinies --> binary vector: root-specific-promoter is hooked to Cas9 gene (for CRISPR) --> transform plants --> Cas9 only expresses in roots and CRISPR only the roots.
(2) transformation 2: Take transgenic plants (w/ single copy transgene) generated from transformation 1--> do a secondary transformation --> also use A. Tumefacinies; binary vector: 35S promoter hooked to your gene-of-interest
Different plant selection marker is need on two vectors. For example, kan for one, and hygromycin for another one.
Make sense to you?
  • asked a question related to Genetic Engineering
Question
38 answers
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.
Relevant answer
Answer
The Sarbecoviruses (SARS and SARS-related beta coronaviruses) are a faily diverse clade of coronaviruses. Merbecoviruses (MERS and MERS-related beta coronaviruses) are another clade with similar diversity. The SARS-CoV-1 and SARS-CoV-2 sarbecoviruses are quite distant from each other.
  • asked a question related to Genetic Engineering
Question
34 answers
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.
Relevant answer
Answer
A paradigm yet. I'm following
  • asked a question related to Genetic Engineering
Question
4 answers
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
Relevant answer
Answer
I guess your are referring to the loxP-flanked transcriptional termination (Lox-Stop-Lox; LSL) element. Here is just one Paper that may be a good starting point for you (Chu et al. BMC Biotechnology (2016) 16:4 DOI 10.1186/s12896-016-0234-4).
There are many plasmids with LSL elements at Addgene.org
  • asked a question related to Genetic Engineering
Question
10 answers
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).
Relevant answer
Answer
Hey Mohammed Shaker Hossain; After conducting all the necessary trials which involve Stage I, Stage II, Preliminary Yield, advanced Yield Trials, Multi-location trials for the target traits of interests, You need data on DUS which is Distinctness, Uniformity and Stability for this variety and it is supposed to be different from the existing varieties that have been released before. You can even go further to genotype it to develop it's fingerprint for reference in case you want to do a QC/QA.
  • asked a question related to Genetic Engineering
Question
2 answers
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
Relevant answer
Answer
  • asked a question related to Genetic Engineering
Question
6 answers
Metagenomics is the study of genetic material recovered directly from environmental samples. The broad field may also be referred to as environmental genomics, ecogenomics or community genomics.
While traditional microbiology and microbial genome sequencing and genomics rely upon cultivated clonal cultures, early environmental gene sequencing cloned specific genes (often the 16S rRNA gene) to produce a profile of diversity in a natural sample. Such work revealed that the vast majority of microbial biodiversity had been missed by cultivation-based methods.
Because of its ability to reveal the previously hidden diversity of microscopic life, metagenomics offers a powerful lens for viewing the microbial world that has the potential to revolutionize understanding of the entire living world. As the price of DNA sequencing continues to fall, metagenomics now allows microbial ecology to be investigated at a much greater scale and detail than before. Recent studies use either "shotgun" or PCR directed sequencing to get largely unbiased samples of all genes from all the members of the sampled communities.
Relevant answer
Answer
Dear Mohammed Shaker Hossain ,
I have attached some books about Metagenomics. I hope they would be useful.
  • asked a question related to Genetic Engineering
Question
2 answers
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.
Relevant answer
Answer
Based on my understanding of the gRNA designing and the related stuff, truncated gRNA are the ones which are devoid of nearly 3 nucleotides (17nt gRNA) in the 5` end. Many studies have shown that excluding the initial 3 nucleotides from the gRNA will increase its specificity without compromising its indel efficiency. On the other side, the last three to five nucleotides at 3' end of gRNA are called as seed regions. Particularly the last 3 nucleotides of the gRNA should not be paired/complementary to the 51–53 nucleotides of the tracrRNA. If it is paired, the sgRNA will lose its ability to create ds breaks or might show less efficiency.
Please read this one to understand more
  • asked a question related to Genetic Engineering
Question
2 answers
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,
Relevant answer
Answer
Hello Sian,
Thank you for the response. We ended up not using any enhancers and got really good integration with some of our targets. However, we do have a couple of constructs/sites that still have low integration rates, so I would be interested in trying out SCR-7 to see if that helps. Do you mind if I ask what concentration you used and/or what procedure you used?
  • asked a question related to Genetic Engineering
Question
3 answers
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.
Relevant answer
Answer
please, see the link below may be useful for you.
Article Construction of a bacterial surface display system based on ..
  • asked a question related to Genetic Engineering
Question
6 answers
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?
Relevant answer
Answer
  • asked a question related to Genetic Engineering
Question
5 answers
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.
Relevant answer
Answer
Probably not. You can do the math. How many repressor molecules per cell? How many cells in the culture? And what molarity of inducer? The result is going to be that inducer is not going to be substantially depleted by binding to all the available repressor molecules.
You may run out of repressors however. Each promoter needs one repressor bound to it, or else it will be active. If you have a high copy number plasmid, you may be absorbing all available repressor proteins from the cell, leaving some promoters unrepressed, regardless of inducer concentration. So you should probably look at provinding extra repressor molecules on your plasmid.
I don't understand why you need them to be independent. If they're induced together, what difference does it make? However, if it really does make a difference for you, you'll need to consider read-through transcription from your other promoters on the plasmid. With 2 independent genes, you would solve that by facing them away from each other. With 3 you can't do that. So you'll need termination sequences between them. I guess it's solvable, but probably takes a lot of testing to make sure everything works the way you imagine.
  • asked a question related to Genetic Engineering
Question
5 answers
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.
Relevant answer
Answer
Andrew Jenkins Andrei S. Babenka Ali Javadmanesh Andrew Ting Thank you very much for your replies and valuable suggestions. A lot of things are cleared now.
  • asked a question related to Genetic Engineering
Question
4 answers
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.
Relevant answer
Answer
Thank you, everyone, for your valuable suggestions. I have seen in a few research papers that people developed tumours without using estradiol supplements. Is it possible to do so? TIA.
  • asked a question related to Genetic Engineering
Question
5 answers
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?
Relevant answer
Answer
Hello, depends what accuracy you looking for. The classical microbiological decimal dilution method will provide more accuracy than Q-PCR for CFU/ml detection.
See info attached may be useful for you.
Good luck!
  • asked a question related to Genetic Engineering
Question
3 answers
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.
Relevant answer
Answer
Then not. I thought you are dealing with inversion.
  • asked a question related to Genetic Engineering
Question
4 answers
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
Relevant answer
Answer
Simple question, but did you design all of your gblocks with the appropriate overlapping overhangs?
  • asked a question related to Genetic Engineering
Question
3 answers
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.
Relevant answer
Answer
Go ahaed and don't worry! As already stated E. coli strains used for cloning are specifically engineered to be RecA deficient.
  • asked a question related to Genetic Engineering
Question
3 answers
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?
Relevant answer
Answer
You can read the following recently publish review article, where you could get your answer as yes:
Application of the CRISPR/Cas system for genome editing in microalgae https://link.springer.com/article/10.1007%2Fs00253-019-09726-x
  • asked a question related to Genetic Engineering
Question
5 answers
I have tried searching everywhere but did not find the source. SspI cuts at AAT^ATT
  • asked a question related to Genetic Engineering
Question
3 answers
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.
Relevant answer
Answer
You may need to check you strain background, S288c strains are gal2, will not utilize or respond to galactose. Switch to a different strains if that's the case.
  • asked a question related to Genetic Engineering
Question
14 answers
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 :)
Relevant answer
  • asked a question related to Genetic Engineering
Question
6 answers
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!
Relevant answer
Answer
I think many of the respondents are mixing up the difference between positive and negative regulation, which is defined as the behavior of the regulatory protein, which is not the same as a inducible or co-repressible system. For example in E. coli both the arabinose operon and the lactose operon are inducible by the sugar, but lac operon is primarily controlled by a repressor and hence negative regulation whereas ara operon is positively regulated by a transcriptional activator (I realize that is an oversimplification for both but primarily correct).
For controlling gene expression and inducible system is normally easier to regulate because you just need to add the small molecule inducer to turn on gene expression, whereas in a co-repressible system you would need to remove the co-repressor (frequently an amino acid or something similar). So from that context inducible is much easier.
Whereas the difference between a negatively regulated vs a positively regulated system is about the behavior of the regulatory protein. The problem with a negatively regulated system is that for controlling a multi copy plasmid you need to have excess repressor present, and usually a single copy of the repressor gene on the chromosome does not suffice. So often negatively regulated promoters are not as tightly regulated and may have substantial basal expression. Whereas for positive regulation you don't have the same difficulty and the basal uninduced expression levels are usually lower (more tightly regulated).
As to why negative regulation is more common in plasmid systems, I think it primarily is historical. The lac operon was extremely well studied and understood in E. coli at the time many cloning and expression plasmids were being developed and it has just stayed with us.
  • asked a question related to Genetic Engineering
Question
7 answers
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 ?
Relevant answer
Answer
It can be any vector of your choice, but I recommend to go for targeted expression in plant system (transient expression in N.benthamiana) could be helpful, If you are not able to achieve expression in E.coli
  • asked a question related to Genetic Engineering
Question
2 answers
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
Relevant answer
Answer
No, sorry, I haven't. Hope you find the bug!
  • asked a question related to Genetic Engineering
Question
3 answers
Qiagen launched their NGS platform GeneReader in Nov 2015, i am curious if anyone can allready comment on it.
Relevant answer
Answer
Hi, we are using Gene Reader for FFPE samples sequencing with very good results.
  • asked a question related to Genetic Engineering
Question
6 answers
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
Relevant answer
Answer
Dear Mohsen, because this expression vector normally is used by gateway cloning approach by which you should first introduce your insert in an intry clone containing GCCGGC . This is the manual for this cloning defined by the company. However using different methods ( that you are also using) are optional and I dont think it makes any trouble for you.
  • asked a question related to Genetic Engineering
Question
8 answers
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?
Relevant answer
Answer
I suppose the smaller plasmids are the plasmids present in DH5 alpha cells for antibiotic resistance. Best cells for transformation, are XL-gold cells, that may accommodate larger plasmids also. I never failed in any of transformation with XL gold cells from Agilent.
  • asked a question related to Genetic Engineering
Question
4 answers
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.
Relevant answer
آسف السؤال ليس من اختصاصي
  • asked a question related to Genetic Engineering
Question
5 answers
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
Relevant answer
Answer
I agree with Rob Keller's suggestion. While the Gaussia signal sequence might work in E. coli, it likely will not be as efficient as an E.coli optimized signal. Why not use an expression vector specifically designed for secretion to the periplasm, there are many of those around. It would be using the universal Sec system if you are utilizing an N-terminal cleavable signal. .
  • asked a question related to Genetic Engineering
Question
4 answers
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!
Relevant answer
Answer
Hi Erwin, ITR is just functional when packaging AAV. If you just want to express a gene in cells, ITR would have no effect, no need to delete it.
You could find more information about AAV on this website: www.genemedi.net/i/aav-packaging
  • asked a question related to Genetic Engineering
Question
3 answers
Is it possible to use gamma irradiation to induce polyploidy on plants?
Relevant answer
Answer
Polyploid also may occur due to abnormal cell division, either during mitosis, or commonly during metaphase I in meiosis. In addition, it can be induced in plants and cell cultures by some chemicals: the best known is colchicine, which can result in chromosome doubling, though its use may have other less obvious consequences as well. So it can be inducted by x ray irradiation as well but you cant exactly control the process.
  • asked a question related to Genetic Engineering
Question
8 answers
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]
Relevant answer
Answer
So you are referring specifically to the STR method (https://en.wikipedia.org/wiki/STR_analysis) "A Short Tandem Repeat (STR) analysis is one of the most useful methods in molecular biology which is used to compare specific loci on DNA from two or more samples. A short tandem repeat is a microsatellite, consisting of a unit of two to thirteen nucleotides repeated hundreds of times in a row on the DNA strand. STR analysis measures the exact number of repeating units. This method differs from restriction fragment length polymorphism analysis (RFLP) since STR analysis does not cut the DNA with restriction enzymes. Instead, probes are attached to desired regions on the DNA, and a polymerase chain reaction (PCR) is employed to discover the lengths of the short tandem repeats."
Basically, the STR method uses primers flanking the STR loci to selectively PCR amplify the STR loci and compare their length. You would therefore have to alter the sites recognized by the PCR primers (highly conserved locations) rather than the STR regions themselves.
The problem is that the CRISPR/CAS9 method is not 100% effective, in the best published cases up to 86% ( ).
As a result, you would still have around 2% of the pairs of primer sites producing the correct PCR product, which could be compensated by 6 additional PCR cycles.
  • asked a question related to Genetic Engineering
Question
10 answers
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?
Relevant answer
Answer
Barry: Since Nature jumbles genes constantly, there should be no prohibition to our adding our contribution via sound science. My only hesitation over GMO is that corporations might reduce basic foods to tasteless meals! Making the flavor better and the visuals more pleasing is a BIG OK with me!
  • asked a question related to Genetic Engineering
Question
1 answer
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 :)
Relevant answer
Answer
Hi, Karmon
I hope you are well, I think trying to KO in adult mouse brain is a very difficult task since its non dividing cells, am not sure that your transduction efficiency will be 100% and your KO cells will be Homozygous.
SO, I think it will be better if you try dCas9 will give you considerable and reliable effects, transduction of your sgRNA targeting your interest gene promoter in dCas9 KI mice
Best of Luck
  • asked a question related to Genetic Engineering
Question
2 answers
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..
Relevant answer
Answer
There are different ways CNVs can be detected or validated, but the method you choose will depend on what you are trying to find.
- If you want to detect CNVs in multiple genes or regions of a genome (other than by WGS) you can use arrayCGH (aCGH, as mentioned above) or by SNP arrays such as (Affymetrix SNP 6.0 or CytoScanHD)
- If you want to detect CNV in a specific gene then  real time PCR (qPCR, as mentioned above) or digital droplet PCR (ddPCR) approach could be used.
in my opinion, i can suggest you to use DDPCR, Digital PCR, an end-point PCR method, does not rely on assumptions of perfect amplification efficiency or on reference standards to reach high precision in copy number enumeration.
for the statistical part it's better to used R software and specially you can use CNVtools ( A package to test genetic association with CNV data ) or matlab bioinformatic toolbox.
for the protocol and how to use you can read this protocol
Chapter Copy Number Variation Analysis by Droplet Digital PCR
  • asked a question related to Genetic Engineering
Question
6 answers
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
Relevant answer
Answer
So your goal, if I understand correctly, is to create a strain of bacteria that continually express a transgene? If so, maybe using a plasmid is not the best option. You might want to consider a stable integration in the genome e.g. by lamda-red recombination. Selecting the transformants will be very easy as colonies should be luminescent. Also consider using a selective mechanism (e.g. toxin-antitoxin or something similar) to prevent gene inactivation on the long term.
Hope that helps
  • asked a question related to Genetic Engineering
Question
4 answers
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?
Relevant answer
Answer
Basically what Artur said:
Be sure to have the original bacterial stock just in case something goes wrong. From this original stock generate one ore more secondary stocks which you will actively use.
Additionally, keep an isolated plasmid stock as secondary fall-back option, so you can potentially transform a fresh E. coli stock (or even another species/strain for other uses).
Also remember mutation rates leading to spontaneous resistances are extremely low, so it is unlikely you will encounter this problem. As mentioned by Artur a loss of plasmid or loss of function are far more likely.
It is also recommendable to renew your agar plate cultures regularly from your frozen "secondary" stock. This is not only to decrease the chances of mutations, but also since the bacteria will adapt to growth on agar after some time, increasing lag time and potentially decreasing growth rate in liquid cultures.
Finally, when producing a new "secondary" stock it is always a good idea to isolate the plasmid from the same culture you used to generate the stock in order to make sure the plasmid is still present.
  • asked a question related to Genetic Engineering
Question
4 answers
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?
Relevant answer
Answer
It sounds absurd. If you grow the strain containing plasmid without selection pressure, you may get recombinants, only strains without plasmid or other contaminants along with the colonies containing the plasmid. Then it will be very difficult to isolate the plasmid. The only way to conserve the plasmid in E.coli is to put selection pressure. Perhaps, you can grow the strain in broth containing ampicillin and isolate the plasmid from it for storage. More over, it is not a good idea to integrate the plasmid in bacterial dna, since it will be difficult to integrate and it will lead to the overall change in the bacterial genome and lot of other difficulties following.
  • asked a question related to Genetic Engineering
Question
3 answers
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
Relevant answer
Answer
Just a bit of an addition, if you wish to preserve the above strain of E. coli with that particular plasmid, there is no other way to prevent the plasmid loss or plasmid curing unless you keep the selection pressure on the strain.However, there is an alternative although not very preferable, what you can do is culture the first primary culture in the presence of the antibiotic and make several glycerol stocks of it (the number depends upon the number of experiments you wish to carry with no antibiotics). Once you have made the glycerol stocks, you may use the fresh set of bacteria every time you need it. But do bear in mind that this increases the chances of not only extra contamination in the culture as there would be no selective advantage for the bacteria containing the plasmid since the strain without the plasmid would grow faster than the strain carrying the plasmid but also the chance of having mix culture of bacteria as in those with the plasmid and those without the plasmid, the only difference here is that the contamination rose from existing strain of bacteria. Hence, unfortunately, unless you modify the bacterial strain or change the characteristics of plasmid, I find it slim chance of preserving the plasmid without the selection pressure..
  • asked a question related to Genetic Engineering
Question
2 answers
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?
Relevant answer
Answer
Dear Emmanuel
In a study to establish callus production in Alternanthera sessilis grown under different combinations of growth regulators and light qualities and to assess whether these factors can increase betalain and flavonoid production, the most suitable treatment for callus formation and subsequent betalain and flavonoid induction was to combine a medium containing 6.7 μmol L⁻¹ 2,4-D and 9.0 μmol L⁻¹ BAP and blue light.
  • asked a question related to Genetic Engineering
Question
28 answers
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.
Relevant answer
Answer
There are linkers based on the same principle than the (GGGGS)n which I find superior as they don't have repeats that can lead to homologous recombination. One example: GSAGSAAGSGEF.
ref for the GSAGSAAGSGEF liner : Waldo, G.S., Standish, B.M., Berendzen, J., and Terwilliger, T.C. (1999). Rapid protein-folding assay using green fluorescent protein. Nat. Biotechnol. 17, 691–695.
ref for other similar linkers: Chen, X., Zaro, J.L., and Shen, W.-C. (2013). Fusion protein linkers: property, design and functionality. Adv. Drug Deliv. Rev. 65, 1357–1369.