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1 + Enzyme (Selecthre) -m- Enzyme (Nonse1ective)l Figure 4.2û. Cornparison of Cell Mass and Emyme Concentration between Seiective and Nomelective Media during Coatlmious Culture in ALR (D = 0.10 h", W M = 1.0, T = JO O C and Go= 10 L).
5.1 Introduction CHAPTER 5 MODELING OF RECOMBINANT S. CEREVISIAE Many genetic and environmental factors must be considend in developing a recombinant cell bioprocess. Quantitative rnodeling of nxombinant cells is very useful for organizing the iiterature and experirnental information, understanding the nature of recombinants. and design, scale-up, operation, control and optimization of recombinant bioprocesses. In this chapter. a metabolic mode1 describing ceii growth. substrate consumption, ncombinant protein production and plasmid segregation of recombinant yeast is proposd by utiüzing the information obtained in this work and the avaiiable knowledge in the üteranue. Although the model's simplinty and feasibility for engineering applications are emphasized, the key metabolic features d yeast are still incorporatecl into the modeL Experimental results in both batch and continuous fermentations of the recombinant S. cerevisiae C468IpGAC9. are w d to evaluate the accuracy and feasibility of the model m descnbing the obselved phenomena To test the generality of the model, verification k ais0 performed with sets of experimental data hm independent studies for other recombinant yeast saains.
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BIOREACTOR STUDIES OF HETEROLOGOUS
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nie University of Waterloo requin%
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ate depended on activities of the p
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Acknowiedgments Fmt of aü, 1 am ve
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2.6. Concluding Rernarks ..........
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5.3.3. Simlilated Results for Prese
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Table 1.1. Table 1.2. Table 1.3. Ta
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Figure 2.1. Figure 3.1. Figure 3.2.
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during Continuous Culture in Airlif
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Figure 5.3. Comparison between Mode
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Cell Systern at Glucose Feed Concen
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a .......... growth ratio (dimensio
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Y, ... ethanol yield for fermentati
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foreign plasmids. One major chalien
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than the denanired, inclusion body
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Table 1.2. Strategies for Enhancing
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Table 1.3. Host Cek, Pfasmids, Gene
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1.4. Immo bilized-Cell Bioreactors
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1. Study the gmwth characteristics,
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plasmid are used; or chromaromal DN
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2.2.1. Genetic Factors 2.2.1.1. Pid
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1- fkquently than a plasmid with Io
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Da Silva and Bailey (1991) used the
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LEU2 or TRPI is cloned into the aux
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eported reduced stabiliity in the s
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on metabolic pathways. In the ferme
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OC), oniy about 35% of the populati
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loss @) is dehned as the probabilit
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+ s Dilution Rate = p' = CI,- - K*+
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concentration couid Iead to coexist
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that factors other than immobiiizat
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earing and plasmid-tke =Ils. Gel be
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productivity irrespective of the mo
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inactive and useless. The problern
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Aerobic fermentation osing iamnobih
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(ranghg h m 1 to 7) on citric acid
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celi fraction decreases monotonicai
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(1987) model aliows for plasmid ins
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affect plasmid stability have ben o
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CEtAPTER 3 MATERIALS AND METHODS 3.
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stiU deleted hm the plasmid. Such p
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1) Reagent solution preparation was
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nonse1ective agar plates, but only
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working Liquid volume. The aeration
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3.5. UNnobilized Ceil Culture 35.1.
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3.52 Cd Lmmobiiization Yeast attach
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3.6. Experimental Error and Reprodu
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-0-Cell(1) +Total CeII (2) 4- Ethan
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* . I I a O 2 4 6 8 10 12 14 Batche
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Cell Maso, Glucose and EthanoI(g1L)
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4.2.2. Batch CuIture in Stirreà-Ta
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[t ~lucoamylase (ALR) -e Glucoamyla
Page 105 and 106: The finai giucoamyiase concentratio
Page 107 and 108: Bentley and Kompala, 1989; Satyagal
Page 109 and 110: 160 First ExponenUaI Phase 1 -c ALR
Page 111 and 112: Figure 4hl. Pathways of Yeast Intem
Page 113 and 114: fermentation (using nitrogen spargi
Page 115 and 116: 43. Continuous Suspension Culture C
Page 117 and 118: Figure 4.9. Continuous Suspension C
Page 119 and 120: 1 +Enzyme +Fradlan of PBC +CeIl Mas
Page 121 and 122: Figures 4.12 and 4.13 show that by
Page 123 and 124: Fi- 4.13. Cornparison of Glu~iase C
Page 125 and 126: where 6 = p- - p' + pp*, which is a
Page 127 and 128: Figure 4-14. Effeds of Dilution Rat
Page 129 and 130: Impoolsup et ai. (1989a) and Hardji
Page 131 and 132: Figure 4.16. dB/dt versus B Plots a
Page 133 and 134: Figure 4.17. Totai Cell Mas at Diff
Page 135 and 136: another recombinant yeast (impoolsu
Page 137 and 138: other recombinant yeast under nonse
Page 139 and 140: 4.3.4. Effixt of Dilution Rate On R
Page 141 and 142: Figure 4.18- Etlects of Mluiioa Rat
Page 143 and 144: Number of Generations - - - - - - *
Page 145 and 146: B Table 4.5. Metabolic Pathway Anal
Page 147 and 148: concentration. But this may be more
Page 149 and 150: [+ Enzyme (ALR) + Enzyme (STR) +Fra
Page 151 and 152: necessary for host ce& to grow in t
Page 153 and 154: Glucoamylase ConcentraUon (unltsll)
Page 155: [a Selective Medium A Nonseiective
Page 159 and 160: handle. excess glucose is femented
Page 161 and 162: The carbon fluxes h m the three met
Page 163 and 164: 53. Modei Simulation in Batch CuItu
Page 165 and 166: Table 5.1. Summary of Parameters fo
Page 167 and 168: 1 n Glucose o Cell Mass O Ethano1 A
Page 169 and 170: detennined in the cunent study, the
Page 171 and 172: Figure 5.4. Cornparison between Mod
Page 173 and 174: equations are üsted in Appendix G.
Page 175 and 176: a dilution rate at which the giucoa
Page 177 and 178: [ o Expetfmental data -Simuiated mt
Page 179 and 180: The effixts of dilution rate on the
Page 181 and 182: Ttme (hrs) 1 a Enzyme A Phsrnid-Bea
Page 183 and 184: It may take several generations for
Page 185 and 186: understanding of the nature of the
Page 187 and 188: On the other hand. the net accumula
Page 189 and 190: Differentiating Equation (6.20) res
Page 191 and 192: Enyme collcentration m the t>ioreac
Page 193 and 194: Glucoamylase Concentration (units/L
Page 195 and 196: 50 100 150 200 Time (hrs) 1 O Free
Page 197 and 198: F i 6.6. Cornparison of Fnx Cd Mars
Page 199 and 200: . - O 50 100 150 200 250 nme (hrs)
Page 201 and 202: Nasi et ai.. 1988) and recombinant
Page 203 and 204: estimated by using Equation (6.24)
Page 205 and 206: [a lmmobilked System O Free System
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oxidation pathway. W~th increasing
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O 50 100 150 200 250 300 Tirne (hrs
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epeated batch fermentations, the fe
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Glucoamylase Concentration (unitslL
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6) Suitable for repeated-batch and
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ecause glucose fermentation dominat
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cells. Tests of the proposed model
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7.2. Recommendations The foUowing f
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APPENDIX A: PLASMID MAP FOR pGAC9 l
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where Bi (Biot number) = - ks De @
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APPENDK C. EFFECTS OF INITIAL GLUCO
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Figure C.2. Effect of Initial Gluco
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Figure C.4 Effect of Initial Glucos
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APPENDLX D. COMPARISON OF ORIGINAL
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(continue)
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(continue)
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(continue)
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E.2. Batch Fermentation with Recomb
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(continue)
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(continue) tfhJ 0.5 10.5 12.75 12.5
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(continue)
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E.3.2. Experimental Data (Parker an
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Run No: CALE (Selected Strain. D =
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Run No: CALR 5 (Original Suain, D =
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Run No: CSTR3 (Selected Suain, D =
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(continue)
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Run No: CICR2 (Selected Suain, D =
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Run No: CiCR4 (Selected Suain, D =
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Eh. Repeated Batch Culture in hmobi
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APPENDIX F. C CODE FOR THE PROPOSED
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* the initial data */ /* gening the
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* getting the founh term in R-K met
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I*Plasrnid-bearing ceU concentratio
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* Func tion C 1 */ îloat C l(float
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APPEMIM G. EXAMPLES OF MAPLE V CODE
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REFERENCES Ataai. MM. and Shuler. M
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Caunt, P.. hpoolsup, A.. and Greenf
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cuitanes of E. coli BZ18@TG 201) wi
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Gabrieisen. O.S. et aL (1990), Effi
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Huang, C-T., Peretti, S.W., and Bry
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Kim, B.G. and Shuler, M.L. (1990a),
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Lee, S.B.. Ryu, D.D.Y., seigel, R,
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Nasri, M., Sayadi. S.. Barbotin, J.
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Pin, S.J. and Kmwski. W.M. (1970),
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Seo, I-H. and Bailey. J.E. (1985).
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Tumer. B.G., Avgerinos G-C.? Melnic
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Yu, J. and Tang, X (Editor, 1991),
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