Bacterial Production Lab

Transcription

1 acterial Production Lab Concentration State variables and processes U DOM State Var. Process r : acterial Growth Rate (gc h - ) CO 2 Other compounds (e.g., EtOH) Objective: Measure bacterial growth rate (also called bacteria production) Why do we want to measure processes? Turnover: []/r DOM G G DOM DIN DIN Time

2 Growth Equation t d Where: t t 0 x t = x t 0 2 t d t d Doubling time of population. x(t) Number or mass of cells per unit volume at time t. Note, cell mass or numbers are easily converted if we assume cells are all the same size: x(t) = n(t), where is the mass per cell and n is the number of cells per unit volume and x(t) is the mass of cells per unit volume. Specific growth rate, Take derivative of above equation with respect to time. r = dx t dt = μx(t) ln( 2 ) ; t d Specific growth rate dx ( t ) x ( t ) dt x ( t ) r dx x dt x ( t ) x ( 0 ) e t 2 Doubling rate ; 2 t d Recall: da dt f ( t ) a f ( t ) ln( a ) df ( t ) dt

3 How are growth rates measured? Accumulation or Loss Rates O 2 CO 2 r Isolate bacteria (How?), then measure: r = dx t dt dco 2 dt do 2 dt What is main problem with this technique? Use a Tracer r DOM r lue = lue t [lue 0 ] t CO 2 where r lue is the rate of blue accumulation and f is the fraction of dissolved organic matter (DOM) that is labeled blue. G ; r = r lue f Tracer Requirements Should not change environment Not preferentially consumed. acteria must utilize for growth Must be able to measure at low concentrations. Low detection limits reduce incubation times. Need some measure of f

4 Radio-isotope Tracers Radionuclides typically used in biology: Half Life Type Tritium ( 3 H) 2.26 y Carbon-4 ( 4 C) 5730 y Sulfur-35 ( 35 S) 87.2 d Chlorine-36 ( 36 Cl) 300,000 y Phosphorus-32 ( 32 P) 4.3 d Iodine-3 ( 3 I) 8.06 d, Iodine-25 ( 25 I) 60 d Types Units: Curie, Ci: disintegrations per min (DPM) (activity of gram of radium-226) SI Units: ecquerel, q: DPS = 60 DPM = 2.7 x 0 - Ci Source of He Helium nuclei Electron Gamma ray For bacterial production, 3 H and 4 C used. Note, 3 H and 4 C are weak emitters, so shielding is not required. Specific activity (SA): Ci mmol - Concentration: Ci ml - Radioactivity measurements: Geiger counter Scintillation counter (method we will use) Measurements are given in counts per min. (CPM) Due to some losses, CPM < DPM Levels of detection SA: 37 mci (mmol 4 C) - Measure: 0 CPM~0 DPM Detect: 0-4 mol 0 fmol Annual Limit on 4 C Ingestion: 2 mci

5 Radiation Exposure Limits and Comparisons (UC Davis) rem: Roentgen equivalent man. Sievert (Sv) = 00 rem Dose Equivalent Limits (Monitored Radiation Workers) Targe Tissue Regulatory Limit UC Davis Guideline Whole ody 5000 mrem/year 2500 mrem/year Extremities mrem/year mrem/year Skin of the Whole body mrem/year mrem/year Fetus 500 mrem/gestational period 50 mrem/month Common Radiation Exposures (Natural Sources and Human Made) One Coast to Coast Flight Natural ackground Radiation in the U.S. Chest Radiograph, A/P view Chest Radiograph, Lateral view Screening Mammography (film/screen combination) Computerized Tomography of ody (20 slices) 3 mrem mrem/year 5-25 mrem/view mrem/view mrem/view mrem iologically Significant Radiation Exposures (Absorbed/Acute Exposure) Risk of contracting cancer increased 0.09% Temporary blood count change Permanent sterilization in men Permanent sterilization in women Skin Erythema Cataract formation 000 mrem mrem mrem mrem mrem mrem

6 What Compounds to Label? Can t 4 C-label all DOM, so label only certain compounds Glucose? 000 CPM Glucose 5000 CPM 6000 CPM CO 2 Can measure growth efficiency if CO 2 is captured. What fraction of the bacterial cell is produced from glucose? Glucose DOM CO 2 Glucose CO 2 Glucose Starch Problem: it is difficult to know what fraction of bacterial synthesis comes from glucose. Label macromolecules instead using appropriate monomer: Monomer % CDW Protein Amino Acids 55.0 RNA A, G, C, U 20.5 DNA A, G, C, T 3. Cultured E. coli

7 acterial Production from 4 C-Leucine Uptake Use 4 C-leucine to measure the rate of bacterial protein synthesis. Calculate bacterial production rate using the following pseudo constants : Pseudo constants: Leucine content in protein 7.3 mol % Protein Ave MW 3.9 Protein Cell dry weight (CDW) 63 % CDW 54 % Carbon Isotope Dilution Problem Occurs when radioisotope is mixed with nonradioisotope. Glucose Extracellular Caused by presence of Leu in solution. Leu Concentration is small (< nm), so add >0 nm Leu and ignore extracellular dilution. Intracellular Caused by de novo Leu synthesis. Assume negligible, or measure. Leu Ext Leu I iosyn.

8 Assessing Isotope Dilution Uptake Rate Leucine Incorporated Extracellular dilution: Measure background leucine concentration. Construct kinetic curve (top right fig). Construct time course curve (bottom left fig) Intracellular dilution: Measure Sp. activity of Leu in protein. Measure actual protein synthesis rate and compare isotope-measured value. Often, intracellular dilution is assumed not to be significant Leu Conc. (nm) Incubation time (min)

9 Example Calculations DPM Experimental Setup SA Leu: 00 Ci mmol - Incubations: 5 and 30 min Volume.5 ml Measure activity after incubation 20,657 DPM (from slope) Note, CPM DPM y = 20657x Time (min) Leu DPM min Ci DPM 00 mmol Ci Leu 000 mol mmol Leu Leu mol Leu min Cells mol Leu min mol Protein mol Leu 32 g Protein mol Protein 0.63 g DCW g Protein 0.54 g g C DCW gc min P gc min 440 min d.5 ml 000 ml l gc l d gc l d 0 6 gc gc 2 mol gc C.7 mol l d C

10 Notes Similar procedure can be done using thymidine incorporation into DNA. Centrifugation plus rinsing (or filtration plus washing) is used to separate added Leu from bacterial incorporated Leu. A killed control is run under identical conditions to account for abiotic adsorption of Leu onto particulate matter. Isotope dilution due to extracellular matrix may not be insignificant in eutrophic environments. Conversion factors are dependent on cellular conditions, and values reported are controversial. Often, only Leu incorporation is reported (i.e., not converted into cell biomass).

11 Safety Notes Wear gloves had lab coats at all times No open-top shoes Wear safety glasses (TCA is an acid) Keep all radioactive materials on your trays and conduct lab work on trays Eject pipette tips onto kimwipes on your tray All liquid waste must be pored into jugs labels for 4 C waste All solids that come into contact with 4 C must be disposed of in 4 C solid waste bins If you spill a sample containing 4 C, lets us know so it can be properly cleaned.

12 Sea Level Rise Land Use Change Plum Island Estuary

13 le u c in e i n c o rp o ra tio n (p m o l /l*h ) Example: Isotope Dilution yron Crump L e u c i n e s a t u r a t i o n c u r v e s J u l J u l Au g a d d e d 3 H -le u c in e (n M )

14 L e u c in e i n c o rp o ra tio n (p m o l/ l*h ) Example: Plum Island Estuary Survey (yron Crump) a c te ria l a c tiv ity 4000 J u ly - 2, W h o le w a te r fr e e -liv in g (< u m n ite x ) C o n d u c tiv ity