User:Janet B. Matsen/never forget

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=== Alternate enzyme options ===
=== Alternate enzyme options ===
==== ACS ====
==== ACS ====
-
* E. coli ACS is a bifunctional protein.  We used to use it, but it had stability issues.
+
* E. coli ACS is a bifunctional protein, [http://www.uniprot.org/uniprot/P0A9Q7 adhE].  We used to use it, but it had stability issues.
** Amanda suggested we only used part of the enzyme, and that resulted in stability issues.  
** Amanda suggested we only used part of the enzyme, and that resulted in stability issues.  
==== ADH ====
==== ADH ====

Revision as of 10:15, 8 October 2013

Contents

Math/Stats

Significant Figures

  • If you have a measurement like 12.34, and you are showing the error, would you write
    • (a) 12.34 ± 1.56
    • (b) or do you include the number of significant figures in your measurement: 12.34 ± 1.567?
    • Mary: I would go with (a). That is certainly how all of the journals I publish in handle this.
  • If you are using a lac promoter and you add IPTG, arent you also inducing expression of LacZYA?
    • I think so. But if you have a high copy plasmid, you are making much much more of your enzyme.

Standard deviation vs. standard error


Standard Deviation Standard Error
Formula (standard deviation)/sqrt(n)
Scale with Sampling Doesn't change much with increased sampling Scales by 1/(sqrt(n)), where n is the sample size. (Always smaller than standard deviation!)
Default Use Describe how scattered samples are Describe uncertainty of the estimate of the mean. Calculate confidence interval & P-value in many cases.

Chemicals

  • NH3 = ammonia, NO2 = nitrite, NO3 = nitrate
    • "The preferred nitrogen electron acceptors in order of most to least thermodynamically favorable include nitrate (NO3−), nitrite (NO2−), nitric oxide (NO), nitrous oxide (N2O) finally resulting in the production of dinitrogen (N2) completing the nitrogen cycle." Wikipedia
    • Nitrifying bacteria: ammonia --> nitrite OR nitrite --> nitrate
    • Denitrification = reduction of nitrates back into the largely inert nitrogen gas (N2): NO3− → NO2− → NO + N2O → N2 (g)
      • performed by bacterial species such as Pseudomonas and Clostridium in anaerobic conditions: use the nitrate as an electron acceptor in the place of oxygen during respiration.
    • Some types eat ammonia; they produce nitrites as waste. Others eat nitrites; they produce nitrates as waste.
  • DTT is a reducing agent which will help keep the Cys thiol groups from getting oxidized
  • Tris-HCl and Tris are NOT the same! (2012/11/14)

Biotechnology

Why are bacteriophages a big problem for bacterial biotech, but viruses in mammalian biotech not?

Ans (from dialogue with Mary Lidstrom 8/2012)

  • Phage can be encapsulated in tough capsids. However, viruses infecting multicellular organisms, they evade the immune system by coating themselves with a portion of the cell membrane, but that makes them fragile outside a host.
  • That doesn't happen for yeast, but the yeast viruses seem to be transmitted through the yeast mating process and don't infect the host cells from the outside. I don't know why that would be, except perhaps because it is a safe mechanism for transfer and yeast do mate at a high frequency. That means of course that even if an infection occurs, sterilization to get rid of the infected yeast gets rid of the virus also. That's much less problematic than having to scrub all the ventilation systems, walls, etc. to get rid of phage.

Metrics of yield/success

  • Yield (%), titer (g/L), productivity (g/L/hr) - Eli Groban of Intrexon 2/2013
    • byproduct profile & strain robustness - "From the first drop to the first truckload: commercialization of microbial processes for renewable chemicals" by Stephen Van Dien 2013
  • Also in Van Dien paper:
    • "For production of basic and intermediate chemicals with selling price near $1.00/lb or lower, the raw material cost of sugar represents a significant fraction of the value of the product even at near theoretical yield. Thus target yields generally need to be at least 80% of theoretical yield even be considered for commercialization. In contrast, with a few notable exceptions cells do not direct a high percentage of carbon flux to these compounds, if any at all. Therefore, the metabolic engineer is faced with the challenge of redirecting a major portion of flux away from biomass production and natural fermentation products, and toward the product of interest."
    • "As a rule of thumb, 50 g/L is the minimum acceptable titer for any basic or intermediate chemical, and may be higher in many cases."

Maximum Theoretical Yield Calculations

  • Options:
    • Use FBA
      • example: "Metabolic flux analysis is used to determine the relative flux of all the reactions in a cell while satisfying all cellular constraints of mass, energy, and redox. When the uptake rates are experimentally determined, metabolic flux analysis can be used to determine the maximum production rate, and subsequently yield, of the product of interest."
    • Use chemical formula of dry biomass and the desired product

Desireable metabolic products

  • branched chain alcohols
    • "branched" is key because these compounds' properties include...

Why are yeast a favorite industrial organism?

  • Oxygen transfer is the largest thing to scale in industrial processes, so you want an organism that can grow anaerobically. You don't, however, want an obligate anaerobe because it is so much harder to work with and grow starter cultures of.
    • Example: clostridium naturally produces butanol, but the fact that it is an obligate anaerobe motivated scientists to move its butanol production pathway into E. coli.

Protein Modeling/Fusion Proteins

Can you use a protein structure from a database to preduct whether you can use that protein as part of a fusion protein?

Ans: from 2012_09_11 summary from e-mail from Justin about visualizing FLS & ADH

  • You may have the structure file, but you are going to run into two issues:
    • "First, the floppy ends of the proteins are not in the structures. At the N and C terminal, you will often see a discrepancy between the actual sequence and what you see in a crystal. This is because you can only see parts of the protein that are essentially fixed in space and not moving around much. So where exactly to overlay the proteins won't be totally clear.
    • Second, as long as the ends are floppy you really won't be able to predict how it looks and the units interact. The only reason to look at a structure for fusions is to know if the ends are highly structured and an integral part of the protein (i.e. in a sheet or helix). If this is the case it is best to avoid fusing those sections. However, if the ends are "floppy" or "unstructured" (as in not a helix or sheet, and not an integral part of the protein... this is for sure the case if you can't see the actual ends in the structure) you can most likely make a functional fusion. Now... there may be empirical differences between how well an N vs C terminal function, but their will really be no sound way to predict that. This is generally speaking, there are special cases, such as a protein that ends in a helix and a fusion made starting at a helix so the two proteins are very specifically oriented relative to each other. But these cases are extremely rare."

Basic Biology

How do cells incorporate N from minimal media?

  • reduced nitrogen (NH4+) is assimilated into glutamate and glutamine, then into other nitrogen-containing biomolecules.
    • assimilation of NH4+ into glutamate requires two reactions:
      • glutamine synthetase catalyzes the reaction of glutamate and NH4+ to yield glutamine (in 2 steps)
    • Glutamate is the source of amino groups for most other amino acids, through transamination reactions.
  • If NO3- (nitrate) is provided, they must expend NADH to reduce it to ammonium before it can be assimilated.
    • Some organisms prefer to do this than start with ammonium. See MM1/MM2/MM3/HY

Bacterial Nomenclature

  • From the journal Molecular and Cell Biology: instructions to authors
  • capital 1st letter is for protein product of a gene. + or - mean whether a strain has the WT phenotype
    • (i) Phenotype designations must be employed when mutant loci have not been identified or mapped. They can also be used to identify the protein product of a gene, e.g., the OmpA protein. Phenotype designations generally consist of three-letter symbols; these are not italicized, and the first letter of the symbol is capitalized (e.g., Pol). Wild-type characteristics can be designated with a superscript plus (Pol+), and, when necessary for clarity, negative superscripts (Pol–) can be used to designate mutant characteristics. Lowercase superscript letters may be used to further delineate phenotypes (e.g., Strr for streptomycin resistance). Phenotype designations should be defined.
  • Use lowercase italic letters for genes. You can indicate promoter and terminator sites. The + and - applies at the gene level, too.
    • (ii) Genotype designations are also indicated by a three-letter symbol. In contrast to phenotype designations, genotype designations are lowercase italic (e.g., ara his rps). If several loci govern related functions, these are distinguished by an italicized capital letter following the locus symbol (e.g., araA araB). Mutation sites are distinguished by placing serial isolation numbers (allele numbers) after the locus symbol (e.g., ara-1 hisB5). Promoter, terminator, and operator sites should be indicated as described by Bachmann and Low (Microbiol. Rev. 44:1-56, 1980): e.g., lacZp, lacAt, and lacZo. It is essential in papers reporting the isolation of new mutants that allele numbers be given to the mutations. For Escherichia coli, there is a registry of such numbers: E. coli Genetic Stock Center, (http://cgsc.biology.yale.edu/). For the genus Salmonella, the registry is Salmonella Genetic Stock Centre (http://people.ucalgary.ca/~Kesander).
  • (?) Not sure about 2nd sentence...
    • (iii) Wild-type alleles are indicated with a superscript plus (ara+his+). A superscript minus is not used to indicate a mutant locus; thus, one refers to an ara mutant rather than an ara– strain.
  • There is special notation for the kind of mutation
    • (iv) The use of superscripts with genotypes (other than + to indicate wild-type alleles) should be avoided. Designations indicating amber mutations (Am), temperature-sensitive mutations (Ts), constitutive mutations (Con), cold-sensitive mutations (Cs), and production of a hybrid protein (Hyb) should follow the allele number [e.g., araA230(Am) hisD21(Ts)]. All other such designations of phenotype must be defined at the first occurrence. If superscripts must be used, they must be approved by the editor and defined at the first occurrence in the text.
  • Subscripts/suberscripts distinguish between genetic elements with the same name
    • Subscripts may be used in two situations. Subscripts may be used to distinguish between genes (having the same name) from different organisms or strains; e.g., hisE. coli or hisK-12 for the his gene of E. coli or strain K-12, respectively, may be used to distinguish this gene from the his gene in another species or strain. An abbreviation may also be used if it is explained. Similarly, a subscript can also be used to distinguish between genetic elements that have the same name. For example, the promoters of the gln operon can be designated glnAp1 and glnAp2.
  • Don't refer to a strain by its mutation.
    • (v) Avoid the use of a genotype as a name (e.g., "subsequent use of leuC6 for transduction"). If a strain designation has not been chosen, select an appropriate word combination (e.g., "either strain PA3092 or another strain containing the leuC6 mutation").

Gram + bacteria

  • don't have a cytoplasm. There are gram + methylotrophs, so all of their metabolic rxns are in the cytoplasm.

Fluorescent In Situ Hybridization (FISH)

  • use formaldehyde to fix cells before applying probes. This makes cells stick together, creating the illusion that consortia are tighter than they may be in nature.

Methanotrophy

  • All pure methanotroph cultures are aerobic.  ?? True of methylotrophs, too??
  • Methanotrophs are found at the interface between anoxic and oxygenated environments. Recent evidence points toward the possibility that they ferment some of the formaldehyde produced from methane. The cultures cannot grow in strictly anaerobic conditions.
  •  ?? For a long time, it was thought that the EDD pathway was used to ____, but recent evidence points toward use of the EMP pathway. The EMP pathway is more efficient.
  • All genes for mixed acid fermentation are present.

Mass Spec

Why is it important to correct for natural abundance & pool size in MS experiments? (incomplete)

  • If you see an increase/decrease in the 13C metabolite pool over time, it is possible (or likeley!) that it is due to the total pool size changing. This is called natural abundance correction by Amanda.
  • See Correction of 13C mass isotopomer distributions for natural stable isotope abundance in Mendeley library
    • O and H don't have large natural abundances, but C does:
      • "For elements having low natural abundance of heavy isotopes, such as hydrogen or oxygen, the difference between natural abundance MIDs of unlabeled and labeled fragments is negligible and consideration of natural abundance MID skew is unnecessary. For elements with non-negligible abundances of naturally occurring stable isotopes, such as carbon, the natural abundance MIDs of labeled isotopomers differ significantly from that of the unlabeled ion."
    • They say the best way to correct for natural abundance is to make standards of labled and unlabeled versions of the compounds you are detecting. However, this is costly and often impossible so they propose a mathematical method estimate a correction.

What can internal standard correct for?

  • ion supression
  • metabolite loss/degradation

Types of internal standards

  • 13C, deuterated (2H)

How does ion pairing harm a MS system?

link: Chromatographers frequently have discussed the effect of the ion-pairing reagents on the stationary phase for columns used for ion-pairing chromatography. Apparently, ion-pairing reagents such as octanesulfonic acid (used for cations) and tetraalkylammonium bromide (used for anions) strongly sorb on the surfaces of bonded-silica columns at certain concentrations of organic modifier. The columns become contaminated and cannot be regenerated to their original state, and the story goes that any column used for ionpairing work should be dedicated to that technique and never used again for regular reversed-phase chromatography. Bidlingmeyer (9) disagrees with this generality and feels that the aggressive pH values used for the ion-pairing coupling actually can change the nature of some columns by either hydrolysis of the bonded phase or endcapping silane under acidic conditions (pH 1–3) or by silica dissolution at higher pH values (pH 7–8).

Our FLS pathway

Alternate enzyme options

ACS

  • E. coli ACS is a bifunctional protein, adhE. We used to use it, but it had stability issues.
    • Amanda suggested we only used part of the enzyme, and that resulted in stability issues.

ADH

  • There are several acetaldehyde dehydrogenases.
    • Some are from E. coli metabolism.
      • Ethanol is produced as an anaerobic fermentation product. E. coli sometimes wants to utilize this carbon and energy source once oxygen becomes available. To utilize ethanol, it is converted to acetaldehyde, then acetaldehyde is converted to acetyl-CoA by an ADH. The enzyme in E. coli does both reactions.

DHAK

  • E. coli DHAK is PEP linked
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