Questions related to Microbial Biotechnology
since i am trying to grow deinococcus radiodurans in my lab, i encounter a lot of compititive bacteria like E. coli and Bacillus types. please suggest
I am a Microbial Biotechnology student and I am also interested in Bioinformatics, therefore, I want to know which programming language may help me in Microbial Biotechnology research, although, I know all programming languages have their own benefits, however, which language will be easier to learn for beginner student and may help in significant research work.
I am trying to get CRISPR/Cas9 working in my lab. I have a strain of Aspergillus that expresses the Cas9, and I need a way of introducing the gRNA.
Hi, all. I am now scaling up a GS-CHO cell culture process from lab scale to pilot scale for the first time. I don't know what will happened and what should I adjust. What problems have you encountered and how have you solved them when you scaled up a CHO cell culture process from 1L or 5L stired tank bioreactors to 200L,2000L or larger?such as metabolic flux shift? viable cell density decreased faster? peak viable cell density was higher but titer was low?
When determining titer of T7 phage by plaque assay, I get a gradient of plaques where one side of the plate is clearing and the other half of the plate is a lawn of bacteria. Anyone experience this issue?
I would like to know any simple stain or test bu which i could stain or quantify only the EPS or biofilm present and not bacteria present in it.
Could you please tell about the opportunities available at various Universities and Institutes which offers Post Doctoral Fellowship for Doctoral students of life science stream (Especially Ph. D's of Biotechnology, Microbiology and Biochemistry) from India.
I would like to quantify the OTR and the oxygen demand in different cultures and rpm of shaker by the method of gas in and gas out, but is not very clear which formulas used, and which measurement magnitude are replaces the variables in the formulas.
Normally bacterial cellulose produced from fermentation was in the form of gelatinous layer. Almost 98% of their structure was water. If this gelatinous layer is dry in oven it will become a very thin layer film thus hard to be grind.
it‘s because some of the enzyme（and which one, please？）show no activity in anaerobic condition， or it's because of the energy（NADH/NAD+) Problem？
I met a problem about recombinant protein production from yeast CEN.PK_alpha. The target enzyme should secrete into the medium. First, the strains were cultured in 20 mL medium for around 30 h (OD ~ 0.5) and then they were transferred to 200 mL medium with an initial OD of 0.05. When OD reaches 1, the medium was obtained by centrifugation. After ammonium sulfate precipitation overnight, there is almost no pellet on the bottom.
Is there anything wrong here? Could anyone give me some suggestions?
Thank you very much.
I am new to this field of microbiology. I need someone's help in helping me develop the crystal violet assay in my lab. I am trying to develop a biofilm layer in a drinking water system and I would like to measure the amount. I will use different types of bacteria like E.Coli and S. aureus.
What kind of equipment do I need? A plate reader spectrophotometer? Do I need to transfer the biofilm from the metal surface to a certain plate? Is the absorbance being measured in a liquid phase? How does the reader work?
Dear scientist fellows,
We would like to investigate the effect of various enzyme cocktails on the hydrolysis of a range of biomass and then quantify sugars by HPLC. However we are concerned with microbial contamination. Therefore we are looking for a robust method to prevent any microbial contamination while having minimum effect on the hydrolysis itself. For example, we would like to avoid to autoclave our biomass as it is expected to have strong effect on the polysaccharides.
Our biomass is not sterile (harvested in nature), but has been dried (40°C) and grinded (coffee grinder) and then store at 5°C.
We are thinking of using microbial inhibitor such as Lactrol, Pen Strep, or others. But we don't know which one to use. We are seeking on your advice on that matter !
I tried using Normal LB and YPD still no success. I am conjugation using E.coli S17.1.
I need to produce Oxalate decarboxylase enzyme as a recombinant enzyme. I choosed my source organisim named bacillus subtilis 168 designed my primers (with doubt) and I want to express in pET-SUMO vector. Are there anyone who tried this vector to produce protein for enzymatic reaction ? OR Can you please suggest me a vector system to produce protein for enzymatic reaction ?
I am trying to identify some bacteria at species level and I read gyrB and rpoB are two good genes for that. Please, do you know if there are any universal primers?
I want to know What´s the best method for microbial DNA extraction of banana roots because i need microbial root DNA of banana for metagenomic studies.
I am using a Nanodrop machine to measure cell density at 730 ( Cyanobacterial cell). But I have some doubt that sometimes this machine gives varied data in replication. Does anybody have experience to measure cell density in Nanodrop reliability?
LacZ activity was assayed using a colorimetric assay based on the reaction of LacZ with ONPG (ortho-nitrophenyl--galactoside), with product concentration measured at 420 nm. To allow calculation of specific activities, the amount of protein in samples was determined by the method of Lowry (J. Biol. Chem. 1951, 193: 265-275).
Urgent help please, thanks.
Hi, I am characterizing the transcriptional regulator of Mycobacteirum tuberculosis .
Because we don't have facility for M.tuberculosis culture, I am trying to over express (using PVV16 which has Hsp60 promoter) it in M.smegmatis. but I never got colonies though the clone and vector back bone are fine. So I thought over expression might be toxic to M.smegmatis. so to check its toxicity I did frame shift mutations and found its over expression is not lethal.
However for the same protein Chip-seq data available in MTB Network Portal. By going through their publication in NAR, I realised they over expressed corresponding transcription factors in M.tuberculosis using Tet-inducible system and did Chip-seq analysis.
My question is, is over expression of my protein not toxic to M.tuberculosis but toxic to M.smegmatis or am I simply doing something wrong?
How can I get a over expression strain of M.smegmatis?
I have a basic query, will bacteria express it genes only when the substrate is present, or by normal basic metabolic pathways and other genes controlled by an operon system?
Is this applicable to eukaryotes? If not, what is the condition for expression of a gene in eukaryotes?
In summary, I want to know, whether a gene identified in bacteria by PCR may or may not express phenotypically?
I was wondering whether you can express the following:
CMV Promoter - NeomycinResistance - viral2a - mitochondrial signal peptide - GFP - Terminator.
In theory the 2a will self-cleave and you get a NeoR protein and a mitochondrial tagged GFP.
However, the T2a ("viral 2a") leaves a proline attached to the N-Terminus.
Will this mess up the signal peptide, so the GFP will stay in the cytosol? Or will it still work and be imported into the mitochondria?
The same question is asked for chloroplast targeting peptides, ER localizaton signals, and extracellular secretion signals.
I am trying to obtain axenic cultures from filamentous brown algae. In order to test axenic conditions, i m using agar marine sabouraud (for checking fungi) and agar marine (this is, solidified marine broth; for checking bacteria ) with an incubation time of 2 weeks at 25°C and 28°C, respectively, in the dark. I came with this idea after reading this paper "A new method for removing microflora from macroalgal surfaces" (Klentz et al. 2011). Previous papers in this topic used Zobell 2216 or half-PES with 1% glucose and tryptone as testing mediums under standard conditions (light and temperature according to each macroalgal species).
Should i use Zobell or half-PES medium for texting axenic conditions or the mediums i ve been using are suitable for this?
Thanks in advance
While I am preparing WEB (Water extract molecule from Streptococcus pneumoniae) after 6 hours shaking it supposed to centrifuge in 10000rpm. But my centrifuge machine was shown error that is why I could not centrifuge then. I kept the sample in -20 and after two days I shaked it again in 100rpm and centrifuge for getting bacterial surface protein. My question is after this hampering will I get my require surface protein? Is there any chance to spoil the protein?
I am using mixed culture of LAB and Yeast for cereal fermentation.For enumeration of each bacterium in a mixed culture, I need to add anilin blue to the MRS medium but I didn't found how the different strains will looks like on a plate.
I need to inoculate 50L of media to reach a final concentration of 10^5 CFU/ml. I have a stock culture at 10^7. I need to calculate how much of the stock I need to add and at what concentration the stock needs to be.
if A136 is growing well n LB it mean it is active, then why it is not growing in AT media? can anybody recommend some changes in At media specification?
I have isolated the cellulose degrading bacteria on CMC media, bacteria showed hydrolysis zone, done colony PCR using 16S RNA universal primer and sent the product to compny. the company sent me sequences when I blast on NCBI I got Bacillus stratospheric strain. Now I have to find the gene and want to clone which degrades the cellulose. how can I find?
Hello everyone, I am trying to determine Catalase activity (KatA) in B. subtilis from the exponential phase, cells were exposed to different concentrations of H2O2. However, when I determined catalase activity in the control and induced cells I see differences in the activity. How can this be interpreted just considering that I am working just with KatA? Considering that Catalases generally, being catalytically active enzymes do not follow Michaelis–Menten and follows Bonnichsen-Chance-Theorell (BCT) mechanism.
I am working on Vibrio Parahaemolyticus biofilm , as Vibrio Parahaemolyticus is a pathogenic bacteria. So, i want to detect type III secretion gene .Can anyone help me to suggest some recognize primer with reference for detecting type III secretion gene ?
This question might be a stupid one but I had some argument on it. What I understand is that bacteria can not grow in 10% SDS solution. We generally use SDS to lyse them. Am I right? Some people from my University are claiming that Bacteria or fungus can grow in 10% SDS. Can you guys help me in this regard?
Hello everyone, I am having difficulty growing Listeria monocytogenes biofilms using the Calgary Biofilm Device (CBD) for the purpose of completing a MBEC assay. It seems like the biofilms are not adhering to the pegs, as tested by a CV staining assay. I have tried using different broths (brain heart infused (BHI) broth, BHI broth with 0.1% glucose, BHI broth with 1% glucose, BHI broth with 2% glucose, Tryptic soy broth (TSB) and TSB with 0.6% yeast extract), different incubation temperatures (37 degrees celsius and 30 degrees celsius), different rotation speeds (110 rpm, 200 rpm and stationary) as well as different incubation times (24hrs, 48 hrs, & 72hrs (changing media every 24hrs)). Iv'e tried all of these different methods with no success. Does anyone have any suggestions or different methodology? Thank you!
I was screening phenol utilising bacteria from the fecal isolates using MRS medium... Here I need to compare the isolates with positive control(phenol degrading or resistant strain) and negative control(sensitive strain). So please guide me how to find out the phenol sensitive strain from the identified lab pathogen(E.coli, Staphylococcus sp, Pseudomonas sp, Klebsiella sp, Streptococcus sp, Salmonella typhi)
I don't know, if competent cells and transformation protocols used for E. coli also applied to anaerobic bacteria such as Clostridium acetobutylicum?
I need mass phage production to apply in aquaculture farm (same as phage production for industry application). Does anybody know method or protocol to do this?
Thank you very much.
Mycobacterial curli pili dont have an operon of pili secretion and assembly factors and chaperones. Its a very different picture from other pili types that usually consist of the components of a pathway mapped on one or more operons. This makes it very difficult to design an experiment to discover and characterise these components. Where do I start? Any ideas?
I am expressing some proteases in E. Coli that works under acidic conditions (e.g. pepsin). I am trying to find out if there is a high-throughput way to qualitatively test the activity of my protease mutants on specific protein substrates. For example, after plating my e. coli cells that contain different mutations of pepsin on an agar plate that is made with casein (or other proteins), I would like to be able to see some changes around the colony (it could be a change in color etc.) to qualitatively access the activity of my pepsin mutant. Does anyone know of a way to do something like that?
I have seen papers where people bromocresol green dye on an agar plate to access secreted protease activity. However, my proteases are likely not going to be secreted. So I am also trying to find ways to lyze e. coli cells on an agar plate to release the proteases into the agar.
I am working on the microbial degradation of a herbicide "Metribuzin" and I want to check the difference in protein expression of bacteria in Mineral salt Media (MSM) with higher metribuzin concentration and without metribuzin. Please, anyone guides me what carbon source I can add into the MSM having no Metribuzin for the growth of bacteria...?
I wish to know which microbe is best for Solid state fermentation, fungi or bacteria. What is your suggestions for selection of microbe?
We select thousand of microorganisms on plant composts. We include the microorganisms (Bacteria and fungi) on pellets for the commercialization. The question is, What is the lowest temperature that kill the microorganisms at lag phase? I know that according to the microorganism specy this temperature will change; it s just to have an idea. Thanks
I need to make a solution of phosphatidyl choline (DPPC) to use in simulated lung fluid. I generally dissolve in water for bioaccessibility tests and it never fully dissolves at pH 7.4. I need to make simulated lung fluid to incubate dust microorganisms. I need to filter sterilize the fluid and not autoclave. But I cannot figure out how to make a solution of DPPC without using chloroform or something that will kill microbes. If anyone can help me suggest something to solubilize DPPC (10g/L) so I can filter sterilize using 0.2 micron sterile filters, I would be very grateful.
Currently Im study the biodegradation of polystyrene. When i preparing my control (minimal salt media plate + cell inoculum without carbon source), i observed cell growth on the control plate.
I have the following questions about aeromonas salmonicida¨s growth:
In my lab, actually I´m growing Aeromonas s. in bioreactor (5L VF) with filtered medium BHI (Brain Heart Infusion) + 0,5% v/v yeast extract and 1% NaCl (not autoclaved). For meausure the growth of this bacteria I´m doing growth Kinetics (D.O600 vs time) and we control pH at 6,5 once the culture reaches this value with NaOH 1M (normally the inicial pH before inoculate the bacteria is about 7,3+-0,2), but once the culture reaches 6,5 (after 2 hours approx. with NaOH control) the culture begins to alkalize the medium until 7,1 (peristaltic bomb of NaOH is off when the pH is above 6,5) also in flask assays with the same medium, the bacteria acidific until reach pH 5,8 aprox without pH control or raise in pH).
- May a bacteria like Aeromonas alkalize a medium like this according with the conditions showed up?
- Has someone observed this situation before with this bacteria or with another bacteria ?.
Thanks for you help
Hi guys currently i am doing a subject using crystal violet assay to quantify s.aureus. The negative control is one non-biofilm forming e.coil, verified by sequencing. The absorbance at 600nm is 0.2 for e.coli, and the s.a. is normally 0.8. But after staining, i can still oberve some substances of e.coli around air-surface look like biofilm. I have tried this method for many times and it should be not containmination. Could anyone please tell me what is that? many thx.
When we inoculate any filamentous fungi to any biomaterial for its fermentation first it grow for few days until the substrate is being utilized.I wish to any is there any publication in this line which can give me information regarding the production of Aflatoxin when it is inoculated with any filamentous fungi for its nutrient enrichment.
If I inoculate the E. coli MG1655 in LB and incubate it on bench overnight, will the E. coli do anaerobic respiration or fementation? Since LB is a rich medium with all sorts of metabolites, it may have the electron acceptors for example, nitrate or nitrite. Anybody could help me with this question or direct me to the right literature? Thank you!
I need to develop a culture media that isolates yeast, more specifically Saccharomyces cerevisiae (its okay if grow other yeasts, but if just grow S. cerevisiae would be great) and inhibit the growth of filamentous fungi.
I need to express new unknown phytase-like protein in some host to further study its properties (phytase activity, temp. tolerance etc), but have not much experience in protein expression work. I found paper by Huang (Env. Microbiol. 2011, 13(3), 747-757) in which authors made protein expression in E.coli BL21(DE3), but wonder if this strain would be good candidate because it contains phoA and AppA genes and in theory should produce own phytase which might affect the phytase expression level.
I would be extremely grateful if protein experts can advice me for or against using E.coli BL21(DE3) strain for phytase expression work and/ or recommend other expression strain (E.coli?) which could be used.
Does anybody know, where we can find the sake to prepare the desired medium for Lactobacillus fructivorans ATCC 15435 ?
Is there any substitution medium for this strain !? Can we use the MRS ?
ATCC medium: 142 Lactobacillus sake medium
Yeast extract..........5.0 g
Liver extract..........0.2 g
Distilled water......100.0 ml
Autoclave at 121C for 15 min.
Hi all! We are working on lactic acid bacteria and we want to make the bacteriocin production more efficient. Any suggestions will be very much appreciated. Also, may we ask if you happen to know a protocol (cheapest) on how to isolate and purify bacteriocins?
specially I am looking for mercury resistant bacteria. Will one able to find bacteria with such function (mercury reduction) MerA. Please help. Thanks.
We are running a bioreactor with a chemically defined media (Riesenberg). Is there any downside to growing our starter cultures in a complex media like LB? Would the bugs need to take time adapting to the new environment?
The only way I can think to test is by experimentally trialing LB->RB vs RB ->RB but that would be time consuming.
Is there a microbiologist or someone of that ilk who might know that step in a bit better detail?
PS: We are using LB for shake flasks as it is quick and easy. RB is a bit more involved but good for high density cell growth.
For example, when an inoculum is 5% in terms of v/v, what does the v/v percentage mean?
Also, how is this applied in industry?
The anode compartment contained Shewanella oneidensis, along with Sodium pyruvate, Casien hydrolysate and MSM, I have a basic understanding of how the system works and I require someone to explain what the role of the bacteria is in degrading the dye as well as what the dye breaks down to.
We are about to genome sequence a probiotic strain of Bacillus. For trials, we are using a spontaneous rif mutant (to enable enumeration). Would anyone recommend sequencing both the mutant and original strain to determine what the difference is between the two? For some context - this Bacillus is being prepared for commercialisation, we would be aiming for ~170X coverage, and prob won't close the genome. Any advice would be much appreciated.
I would like to develop a simple method to distinguish antibiofilm surface but with a loss cost and a reduced incubation time.
The surfaces I hace to study are differents, some times are PP, PE and other times are metal surfaces.
i read several standart method like:
-E2196 − 12
Standard Test Method for Quantification of Pseudomonas aeruginosa Biofilm Grown with Medium Shear and Continuous Flow Using Rotating Disk Reactor1
-E2647 − 13
Standard Test Method for Quantification of Pseudomonas aeruginosa Biofilm Grown Using Drip Flow Biofilm Reactor with Low Shear and Continuous Flow1
But in all methods, I have to spend many money and time to build a reactor.
Finally I have decided to do a stactic simple reactor, I am going to use a Falcon (50 ml) with TSB and some micronutrients with a inoculum ( e coli 10^4 UFC/ml) and after I introduce my sample but i an not sure if I have to change the medium every day or how many time i have to incubate the samples?
I would to like to have a homogeneus surface in 72 hours.
First of all let me state that i am not a microbiologist but i have to interprete some microbiological results in water. In our laboratory we use the m-endo les mebrane filter method for the detection of total coliforms and the tbx method for the detection of e. coli. IIs it possible to have more colony count for the e coli than the colony count for total coliforms? eg Total coliforms 10, e coli 28. Thank you very much in advance for your replies!
Could anyone please tell me where I can get the following strains?
AHL degrading strain Pseudomonas fluorescens (P3/pME6863)
AHL non-degrading strain Pseudomonas fluorescens (P3/pME6000)
Agrobacterium tumefaciens AT 136 (pZLR4)
Maybe someone could provide me with a sample?
I study pattern recognition receptors (PRRs) protein in shrimp like this (http://www.sciencedirect.com/science/article/pii/S0145305X1400010X) and (http://www.sciencedirect.com/science/article/pii/S105046481400148X). I suffered a problem in the part of bacteria binding assay and agglutination assay. My recombinant protein can bind to all bacterial (gram-negative, E.coli and Vibrio harveyi, gram-positive, Bacillus megaterium and Staphylococcus haemolyticus and Yeast, Saccharomyces cerevisiae) but cannot agglutinate V. harveyi, S. haemolyticus and S. cerevisiae. I presuppose that binding of my recombinant protein to V. harveyi, S. haemolyticus and S. cerevisiae might activate phagocytosis or encapsulation, agglutinated by my recombinant protein to E.coli and B. megaterium might activate nodule formation.
I need to spike blood with a homogeneous culture of A. brasiliensis to ensure equal quantities of inoculum are present in the PC and the test sample when I aliquot from an in vitro spiked stock.
I have tried various techniques such as increasing the agitation (250rpm), decreasing pH in SD and PD broths (down to pH 2.04), changing temperature conditions (between 28 and 37 C) etc.
I recently tried using the culture to plate and make spores for spore preps in order to increase the level of inoculum...
Has anyone managed to do this or does anyone have any suggestions for what else I could try to either (1) create a homogeneous culture (2) ensure equal inoculum present in PC and test samples?
growing the bacteria(LB broth) for protein expression: the optical density(1.2) , is this good for protein expression(before IPTG).