Microbial Biotechnology - Science topic

Kuhner AG is the best option for measurement, which provides appropriate results

Those are very broad topics. Is this for an entire course or just your lab? Who will be supervising them (you or other colleagues with different areas of expertise)? What are the research themes being carried out in the labs which will be hosting these students?

Can u pls suggest me a selective media for deinococcus isolation

I recommend you R & Python

George Matthews thank you very much!

k_La is a very important design parameter. I have studied the energetic optimization and adaptive control of the dissolved oxygen concentration in aerobic fermenters by numerical simulation. The parameters of the usual K_La correlation were estimated on-line and at real-time through the recursive least squares algorithm with forgetting factor, most effectively when a small sinusoidal disturbance was imposed to the manipulated variables (stirring rate and/or air flow). The power dissipated by agitation was accessed by a torque meter (pilot plant). This investigation was reported at (MSc Thesis):

AS YOU KNOW WELL THE T PHAJES ARE OF TWO TYPES

the ODD. T1, T3, T5 T7, and

THE EVEN ARE T2.T4, T6,

As I KNOW LOT OF WORK AND LITERATURE EXITS BECAUSE THE PLAQUE SIZE ID GOOD ENOUGH TO OBSERVE AND EASY TO WORK AND DETECT MUTATIONS

AND VARIOUS KINDS OF EXPERIMENTs have been done

With odd phages the plaque size is too small and little work has been done

FilmTracer SYPRO Ruby biofilm matrix stain

In Case of China, almost each University has its own provincial postdoctoral fellowship. So getting a proper supervisor is more than enough.

Bacteriocins are proteinaceous toxins produced by bacteria to inhibit the growth of similar or closely related bacterial strain(s). ... Antibiotics or antibacterials are a type of antimicrobial used in the treatment and prevention of bacterial infection. They may either kill or inhibit the growth of bacteria. The differences between them are:

1- Bacteriocins are produced on the surface of ribosomes in microbial cells, while antibiotics are primarily secondary metabolites of the cell.

2- Bacteriocin producers are insusceptible to the bactericidal agents, unlike producers of antibiotics.

3- Bacteriocins can attach to the target cell wall anyplace on the surface, as no specific receptors on the target cell wall apparently exist.

4- The mechanism of bacteriocin on target cells is diverse and is associated with the method of pore formation in the outer cell membrane. Bacteriocins bind to cell walls of sensitive microbes, motive ionic imbalances, and produce pores. Inorganic ions leak the target cells through the created pores and thereby killing them. Antibiotics, on the other hand, can inhibit synthesis of the subcellular processes (cell wall synthesis, intracellular protein production, and DNA and RNA replication). Their bactericidal and bacteriostatic mechanisms are diverse and may comprise pore formation, degradation of cellular DNA, disruption via specific cleavage of 16S rRNA, and blockage of peptidoglycan synthesis.

Hello,

If you want to grow them in rich media, YPD or synthetic wort media work fine. If you are after some physiological characterization in bioreactors where you would like to do carbon balances or other metabolic analysis, then you might go for a more defined media like YNB with maltose or glucose as carbon sources.

With regards,

Rosa

I have tried two methods:

1. freeze dying BC followed by dipping in liquid nitrogen for a few seconds and grinding in a ball mill.

2. freeze dying followed BC by keeping in the refrigerator overnight and grinding in a blender. The powder is sieved and bigger particles are put back in the blender for another round. (modified method of Munair Badshah )

Both of the methods gave BC in powder or particle form.

Agree with Rudy Situmeang

There are (basically) two categories of anaerobic metabolism: (1) fermentation and (2) oxidative phosphorylation that does not use oxygen to make ATP. However, many people are incorrectly taught that fermentation is the only type of anaerobic metabolism. So, when folks say "anaerobic organisms do not have the TCA cycle" what they really mean is "fermentative organisms do not have the TCA cycle". In contrast, it is quite common for non-fermentative anaerobic organisms to use the TCA cycle, such as sulfate reducers, nitrate reducers, iron reducers, etc.

Try this: may be you find some protocols and booklets.

There are several contamination interferences and preventative means that NREL (https://www.nrel.gov/docs/fy15osti/63351.pdf ) have highlighted for enzymatic saccharification of lignocellulosic biomass, please read section 5. Interferences, point 5.9 and 5.10 in attached file. Hope this is of help. Cheers.

Please find below link , may be helpful for KLa be calculated in a flask

https://www.presens.de/support-services/kla-calculator.html

Yes, I know but things have changed a lot since :)

It's much easier now. I can send you the required tools if gene KO is what you are after...

Following

Please have a look at this useful RG link.

I guess so ;)

We have encountered problems with intracellular expression, too. So checking out diffrent strains might be a good idea.

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

Hello, here the global challenges in plant biotechnology.

Increase crop productivity especially in adverse environments,

Management of herbicide tolerance,

Management of resistance to pests,

Management of resistance to diseases,

Improvement of genetic engineering technologies to enhance public perception etc.

What about microbial biotechnology the many topics related with plant biotechnology for example genetic engineering and transgenic crops, biopesticides, bioinsecticides, biofeltilisers, bioleaching, soil desalination etc.

I have utilized CTAB with great success in extracting fungal DNA from leaf litter at a very low cost. The MOBIO powersoil kit has also worked very well with leaf litter.

This document basically suggests that Nanodrop OD600 reads are not accurate for measuring bacterial cell density unless a conversion factor is determined by dividing the cuvette OD to Nanodrop OD, which can then be used in a decently wide range of ODs.

But that also means you need access to a cuvette spectrometer, at least once every time you need to calculate the conversion factor for a specific application/OD range.

Can anyone answer this?

I think everybody so far are giving their opinion but not straight to the point as per the question of Nandagopal. So I again reiterate that yes, some genes are controlled but not all.

I had the same question and found this Cold Spring Harbor protocol/review of 2A peptides, which comments that 2A generally does not affect the localization of downstream proteins except when the upstream protein is targeted to the ER lumen. 

Design and Construction of 2A Peptide-Linked Multicistronic Vectors

Andrea L. Szymczak-Workman, Kate M. Vignali, and Dario A.A. Vignali

Cold Spring Harbor Protoc; 2012; doi:10.1101/pdb.ip067876

Dear José Avila-Peltroche,

I would like to emphasize that my intention was never to discourage your research. Although difficult, it is not impossible to obtain axenic cultures of algae. Good luck in your search.

Thank you very much. I will try.

Dear Khan 

The media uses for lactic acid mix depended on used isolates. Anilin blue with MRS medium is not selective 100%. you can used other media while The yeast  enumeration was used Chloramphenicol glucose yeast extract agar   

If you intent is to establish by inoculation 50 liters at 10^5/ml, think you'll need 500 ml of 10^7/ml inoculum in those 50 liters (i.e. 500ml inoculum in 49.5 liters).  Consider: concentration x volume = concentration x volume.

Check following parameters..

1. composition that inhibit growth.

2. Expiry date of media.

3. Environmental temperature.

4. Contaminating organism.

5. Aerobic / anaerobic conditions.

Read the papers related to this bacteria. and find the probable cellulose, then get that protein sequence on NCBI and then blast together.

or you can read this paper I hope it will help you.

The problem it´s that in the system it´s already overexpressed, I am comparing it in the same OD600, protein has been quantified, and actually when bacteria is exposed to H2O2, population starts decreasing

Dear

This parper can be help you also : http://www.microbiologyresearch.org/docserver/fulltext/micro/155/3/837.pdf?expires=1499462159&id=id&accname=guest&checksum=CFED3D9BF09A6712892977C5A50CFE57

SDS is used in high amounts as a detergent in products such as shampoos, car wash soap and toothpaste. Therefore, its bioremediation by suitable microorganisms is important. I advise you to read the following interesting paper:

Screening of SDS-degrading bacteria from car wash wastewater and study of the alkylsulfatase enzyme activity

Hi:

In order to pellet cells, people usually use around 5000 g (centrifugal acceleration 5000 times the gravity on earth) for 10 minutes.

Regards

Thank you Mert Sudagidan for your valuable input. You are right, this strain of Listeria monocytogenes is probably not a strong biofilm former. We will try with another strain for these experiments, thanks again!

z

i cant find any link down here... 

Thanks James.

Thanks for your answer. This is a very unique protein. When a BLAST search is done on ncbi, the protein does not match any proteins known to assemble into pili in any other pathogens. The protein was characterised by Alteri 2006 upon purification of the pilin subunits by mass spec and was established as curli pili by biochemical characteriastics and appearance of the pili on electron micrographs. What are the steps taken to uncover a secretion and assembly pathways when the genes are unknown?

I agree with Steingrimur Stefansson answers

good luck

You should use a carbon source which directly enters the Krebs cycle, what means, to select one of its intermediates, such as acetate, or citrate. This helps to prevent the induction of additional genes for additional catabolic enzymes (including their transport systems) and, as a consequence, reduces the background for proteomics studies which you have in mind. In this sense, sugars, as mentioned by the colleagues above, are not the first choice.

thank you

Fungi are the most potent microorganisms to grow in solid state fermentation. Filamentous fungi are the best such as Aspergilli, Penicilli, Trichoderma, and so on.

Sidkey, N. M.; Ammar, M.S.; Ashmawy, A.M.; Rady, A.H.& El-Hagar,O. A.(2001): Regulation of the thermo -Amylase productivity by Thermomyce slanuginosus Tsiklinsky, Ferm-Bam,F4 while utilizing water hyacinth as the sole carbon source under solid state fermentation. Al-Azhar Bull. Sci. Vol. 12 (1) June 2001: pp. 243-262.

Younis,N. A.; Sidkey, N. M.; Ammar, M.S.& Ouda, S. M.(2001): Recycling of water hyacinth for production of cellulases by Aspergillus terreus, I5 and Penicilliumm oxalicum, I8 under solid state fermentation. Al-Azhar Bull. Sci. Vol. 12, No. 2, Dec.2001: pp. 353-373.

M.S. Ammar, H.M. Atta, A.T. Abul-Hamd & N. M. Sidkey (2002): Bagasse Recycling For-Lactam Antibiotic Production By Penicillium chrysogenum, Ferm-BAM-1 Under Solid State Fermentation Conditions. Al-Azhar Bull. Sci. Vol.13,No. 1 (June): pp.169-196

Kla depends primarily on power input and medium. Reactor configuration and viscosity may lead to 'dead' zones reducing overall mass transfer. Also oxygen solubilty is important for mass transfer. So measurements are required or someone's else experience in a very similar reactor may give at least an indication.

Thanks you for your answer. My email adress is pblanquet@marcel-mezy-environnement.com

Thank you so much.Appreciate it a lot.

Dear Kiang,

1. Some bacteria can grow without added carbon source. They exhibit an "autotrophic metabolism" and fix carbon in the form of CO2. Traces of CO2 could thus support them.

2. What can also happen is that organic buffers (TRIS, MOPS) are partly degraded as carbon source by some microbes.

3. Finally, chemicals such as salts contain small impurities of organics, depending on their purity. Even water can have some depending on purity. This could also deliver carbon for growth.

4. If you prepare an agar plate, you use a polymer to achieve the solid medium type, which can be degraded by some bacteria. If this is the case, the colonies will sink into the plate becuase they liquify the medium around them. we used this many years ago select for gellan degrading bacteria.

Best,

Christoph

 

Dear Felipe: Do you measure concentration of dissolvbed oxygen? According to my opinion if your culture is limited by oxygen (especially during log phase), it could produce some metabolites which alkalize the growth medium.

Best regards   Vit

thank you all 

What for???

If you recollect your basic organic chemistry, then you will know that CV is no doubt a dye belonging to the pararosaaniline group with a phenolic group which makes it inhibitory to an organism like E. coli. It is always better to use a medium as a negative control rather than any organism. 

Thank you Sir. 

 Thank you both for the answer. I understand that E. coli goes to fermentation if there is no electron acceptor anaerobically. Just to clarify my question, if there is electron acceptor in LB under anaerobic condition? Thanks again!

.

Thank you, Nicole!

 Thank you so much, Mr. Udhia Hussain Jassim Al-Delemi. I will read the articles.

Dear Jameela Esmaeel, I am not sure this medium can be work for Lactobacillus fructivorans ATCC 15435.

Numerous methods are published

Note  Nettles and Barefoot,1993. J Food Protection

Enan etal 1996. Int J Food Microbiol

Hoover 1993

De Vuyst and Vandamme 1992 and others

Dear Ma Reina,

Amplicon sequencing would not be useful due to is frecuently used to analyze diversity using universal genes (16S, ITS). With shotgun you can assess microbial traits with software and databases as GOTTCHA (Genomic Origins Through Taxonomic Challenge) and KEGG (Kyoto Encyclopedia of Genes and Genomes) 

Dear Gabriel

To be more sure that your pre-culture contain an higher % of live bacteria and minimize the lag phase, generally you have to permorm inoculation when the preculture is still in the exponential growth phase or just at the beginning of the stationar.

For reach thisresults in my experience, i used on the basis of the situation 3 different approaches:

1) At 37°C in LB: Do a small preculture O/N from LBplate or glicerol and, use it for inoculate by diluting 1:100-1:50 the real preculture and once it reach an OD around 1 (3-5 h depending from the e.coli strain and plasmid antibiotic resistance) then inoculate your fermenter. OF course this shift all your fermentation timelines and depends if it is compatible with your time work.

2) Replace LB with a medium which allow to reach higher OD (e.g enpresso media of biosilta or also TB) which allow longer exponential phase and therefore after the O/N growth the most of your bacteria are still alive.

3) Use LB and growth it ON but shift down the growth temperature to decrease the bacterial growth rate and reduce the amount of dead cells.

Of course if you would like to be more precise  and carefully evaluate the amount of live and dead cells in your preulture, just the first time during the protocoll set up, you can check the cell viability using some probes (as live dead of thermo e looking it at the fluorescence microscope) but i tihnk that if in LB, you are able to stop your pre-culture  in a good phase ( for example in LB at OD around 1 maximun 1.2) the most of cells will be still viable. 

All this 3 approaches can allow you to minimize dead cells and make the OD a good follow up for the inoculation. 

good luck

Manuele

aerobic condition

This term is very often used once you have standardized your inoculation procedure. For bacteria o yeasts for example, you may count cells and establish that you are going to use a 10^8cells/mL-inoculum, or xx CFU/mL, or a given O.D. inoculum. But then, you need to standardize how much inoculum of this N° or density you are going to add for starting your next culture. Normally, one uses 5-10% v/v, which means what Dr. Belaouni detailed above. If you are setting a1L-culture you may add 50-100 mL of inoculum (or starter). Being strict, quantitatively, you should discount this volume from the total volume of culture (as Belaouni said). In the practice, at pilot plant scale or industrially, many times you directly add this 5-10% without discounting from the whole culture. The main thing is standardization, which assures you the possibility to reproduce your results EVERY time. Other relevant thing is to keep your inoculum, not only in the same proportion, BUT always in the same growth stage and equally activated. In the case of fungi, sometimes the inoculum processing is slightly different. You may neither count (except for conidia) nor measure OD. But you can add the same number of agar plugs per volume, or standardize by dry weight. Hope it serves. Success!

Dear Kumar,

It depends what kind of enzyme activity you wish to study: some (most, probably) might be inhibited by high ammonium sulfate concentrations, other maybe fine. I would recommend that you test both crude extract and dialyzed extract and see if (1) you get any of your desired enzyme activity and (2) if dialysis improve activity. Further purification may be needed, but i would recommend you try that first.

Best regards,

Eric Gomès.

 Thank you for your correcting, Dr. Benedik. I am sorry for imperfect knowledge in my question. Now, I have realized that. On the other hand, I have overcome the problem about my research. Thank you so much again for your consideration.

Purpose:  The glucose tolerance test measures the clearance of an intraperitoneally injected glucose load from the body. It is used to detect disturbances in glucose metabolism that can be linked to human conditions such as diabetes or metabolic syndrome. Animals are fasted for approximately 16 to 18 hours, fasted blood glucose levels are determined before a solution of glucose is administered by intra-peritoneal (IP) injection. Subsequently, the blood glucose level is measured at different time points during the following 2 hours.

 

Scope: 

Minimum number of mutants: 7 mice for each sex

Age at test: 13 weeks

Sex: Both (sexually dimorphic)

Materials:

One Touch® Ultra2 glucose meter

Stainless steel disposable scalpels

Balance

Timer

Glucose solution 20% (0.9 NaCl)

Gauge needle (25 G 5/8)

Syringe 1 ml

One Touch® Ultra Test Strips or Generic Brand

Gauze and alcohol (70%)

Topical anesthetic cream (optional)

Mouse restraining device (optional)

Quality Control

Calibrate the glucometers each day.

Weighing of mice should be completed before the pre-bleed is taken.

Mice should only be housed in a duplex with animals of the same sex.

 

Set-up

Fast mice overnight for 16 hours by transferring mice to clean cages with no food. Ensure that they have access to drinking water at all times

Prepare an experiment record sheet, lab mat work space, syringe, gauze, alcohol, glucose strips, glucose meters and freshly made 20% glucose solution.

Make sure that glucose meter and strips are the same code before starting.

Stick one strip into the glucose meter all the way.

Obtain the Control Solution; check that date of use is before the discard date. If not, discard and acquire a new control. Write the discard date on the control bottle three months from the date opened.

Shake the control bottle before using and put one drop of the control on the glucose strip in the glucose meter. Make sure control solution fills up the strip and touches the base.

Once reading appears compare control reading to the control range on the test strip bottle. Press the right arrow button to log the reading into the glucometer, L1 will appear on the top of the screen, and then press the power button on the side of the meter.

Record the control reading for internal purposes.

Weighing and separating mice

Start weighing the mice in numerical order so that the mice weighed last have some time to calm down before getting a baseline reading.

Grab a mouse out of the cage with deliberate movements trying to reduce any chasing.

Have the cage close enough to the scale that mouse is not suspended by its tail for more than 3 seconds.

Place the mouse on the scale and record its weight. Place the mouse in a duplex by side by itself. The cage should be clean or the clean cage from fasting making sure the mouse has access to water. The mice should be separated so that they are in numerical order. No mouse should be in a duplex with a mouse of the opposite sex.

Once weighing is complete move cages to IPGTT room nicely on a cart. Place cages in numerical order nicely, avoiding any shaking or bumping of the cage.

Procedure

Calculate and record the volume of 20% glucose solution required (2g of glucose/kg body mass) for IP injection as follows: volume of IP glucose injection (µl) = 10 x body weight (g).

Set the test strip in the glucose meter, part of the way. If left in all the way, the meter tends to turn off before blood is administered.

Recap the strip container every time after obtaining a strip. Do not use strips that have been left open to air for extended periods of time.

Clean tail with gauze soaked with 70% alcohol, then dry tail with dry gauze. Make sure tail is dry.

Score the tip of the tail using a fresh or sterilized scalpel blade, only a millimeter or two is needed.

Push test strip all the way into the glucose meter, drop indicator will show up.

Milk the tail and discard the first small drop of blood by dabbing on lab mat.

A small drop of blood (<5µl) from the tail is placed on the glucose test strip in the glucose meter. Make sure blood fills up the strip and touches the base before the 5 second count down ends to get an accurate reading.

Record this baseline glucose fasting level.

Inject the mouse intraperitoneally with the appropriate amount of glucose solution determined earlier by weight and note the time-point of injection on the record sheet.

The blood glucose levels are measured at 15, 30, 60 and 120 minutes after glucose injection, by placing a small drop of blood on a new test strip and recording the measurements. Start the bleeding again by removing the clot from the first incision; massage the tail if blood flow is inadequate from base to tip. Record results on the record sheet.

Ensure that further blood loss from the incision is minimal by briefly applying pressure to the incision after each measurement. At the end of the experiment add food and recovery gel to the cage and make sure water is available to the animals.

Monitor the animals carefully to observe any abnormal behavior(s).

 

Clean-up:

Wipe down countertops, glucose meters, and other materials with Clorox wipes.

Change your gloves between each cage. To save time, double glove for every two cages.

Discard used lab coat due to possible blood contamination.

 

Notes:

Calibrate the glucose meter routinely as outlined in the manual.

Record equipment name, manufacturer, model and serial number each time.

Record if mouse was restrained or not and type of strip used (whole blood glucose).

 

Reference List:

Thank you Dr Alan and Dr Mohamed I appreciate it.

Hi Alan.

We did the same strategy for Streptococcus pneumoniae (for B lactams and quinolones, see our publications ), a new meachnisms were described (ABC, and efflux pumps).

And we did the same for M. tuberculosis vs Rifampin.

In M. tuberculosis, all induced resistant mutants harboured mutations in the small region determining resistance to rifampin. and some mutations are known to be associated with high level resistance. it is also important to see if your mutant are also resistant to other rifamycin drugs.

It will be worthy to seq both strains of Bacillus and see if there is other rearrangements, mutations in efflux pumps or ABC transporters...etc

If you want to close the genome the PacBio is better but more expensive, but for this kind of analysis Illumina Miseq is sufficient to analyse.

To achieve 170X coverage or more, keep in mind that plasmids are also sequenced, and you may need to know how many are present and how much copies ? in some instance rif resistance is also mediated by plasmids.

Dear Lydia, we previously tested biofilm formation ability of our biofilm forming S. epidermidis strain on different ion implanted metal alloy surfaces. We used 24-well plate. The size of our coupons fit these wells. After incubation with bacteria we washed samples and removed bacteria from surfaces and count them. By this way we can compare different surface treatments. 

Another way to use optic microscope after  staining bacteria to determine biofilm formation on metal surfaces, however, to compare the results a little bit difficult.

Best regards.

Hello,

E. coli is a Coliform. So it seems unusual to have more E. coli than Coliforms.

But it is possible by using different media.

TBX is optimized to the growth of E. coli. If you make a dilution and you add the same inoculum to both media you will have more E. coli on TBX than on ENDO

If you want to do more water tests, I would recommend the Chromogenic Coliform Agar according to the ISO 9308-1. There you get both coliforms and E. coli on one Plate in different colure and you will not have these difficulties by interpreting the results.

my dear, 

i sorry, I suggest you to contact the people who worked with these bacteria.

Chapter 1: The immune response to infection

Dear Ali

A four step solvent transfer method was used for the extraction of penicillin from different shake flask cultivation systems. Briefly, first penicillin produced in shake flask culture was extracted in to amyl acetate and then transferred from amyl acetate into phosphate buffer. In third step extraction was made from buffer solution in to chloroform and finally transferred from chloroform in to water. The extracted material was soaked in to sterilized filter paper discs for quantitative analysis.

You can search these, it may help

European journal of applied microbiology and biotechnology

September 1980, Volume 9, Issue 3, pp 199–209

Medical Sciences and Public Health

Vol. 1, No. 1 (2013), 1–5

Brazilian Journal of Microbiology (2012): 

I definitely agree with Truc here, you want to find the right balance between the number of cells and the amount of protein yield you get at the end after IPTG induction. You can sometimes get a higher protein yield when induced at a slightly lower OD. It all just requires some optimisation. 

Hi!

We use Escherichia coli JW5433.

Very simple and practical solution and that is do not use any organic growth medium, instead use medium like M9 medium with say 0.05% yeast extract and dextrose at 0.5%.