11.8 Growth Kinetics With Plasmid Instability A potential problem in culture of recombinant organisms is plasmid loss orinactivation. Plasmid instability occurs in indi- vidual cells which, by reproducing, can generate a large plasmid-free population in the reactor and reduce the overall rate of synthesis of plasmid-encoded products. Plasmid instab- ility occurs as a result of DNA mutation or defective plasmid segregation. For segregational stability, the total number of plasmids present in the culture must double once per genera- tion, and the plasmid copies must be equally distributed between mother and daughter cells.Aime model hasbeen developed for batch cua co deseribe changes in the Faction of pasidceaing ell 4 function atime [12] The important paramecersn tis model auc the probubility of plasmid lace pe generation of cel and thediffeence inde growah sof plasmid beaingand pls- ‘mide cell Exponential growth ofthe ox cels ase Tf isthe concentration of pasidearying cell and x” is the concentration of plasmid ely he rates at which che ‘wocel populations grow re Onpee as and Rotate (1162) ‘where re isthe ae of growth ofthe plasmid baring popula: tins res the ae of growth ofthe plasmic popaation, p is he probability of plasmid los pet ell vision (p= 1). is the specific growth ateofpamid-arying cll, and isthe specif gow rate of plasmid fee cel. The model asumes ‘tha all plasmid-containing cell are identical in growth rate and probability of plaid los this isthe same as ssming that al plasnid-coneaining cells have the same copy number By comparing Eq. (11.61) with Eg. (11.52) we can see hat the sate of powth ofthe plasmid-bering populations reduced by 4 x This isbecaut some ofthe progeny of plsmid-bear ing ces donot contain plaid and do not join the plismid-bearing population. On the other hand, grounh of the plasmid fee population has two conebutons a indica- ced in Eq, (11.62), Exiting plasmid free elle grow with specie growth rate as sua in addition, this population is supplemenedby generation of plaice cls wo dele tiv plasmid segregation by plamid-carrying cel ‘Aan ime, the fraction oeelsn the culture with plasmid Fee c.63) In busch culture where rs of growth can be determined by ‘monitoring ell concentration, ge #)y andy = Ty “Therefore, Eqs (11.61) and (11.62) can Be ied dle taneously with inital condition x” = 3% and x Aer n generations of plasmid-contaning cells: Irony anrerty c160) where aot ¥ (16) and (11.66) ‘The value of Fepends on a, the rato of che speifie growth rates of plasmid-free and plasmid-cartying cells. In che absence of selection pressure, presence of plasmid usually reduces the growth rate of organisms due tothe additional metabolic requirements imposed by the plasmid DNA, Therefore aris usually > 1. In general, the difference berween Mf and ye becomes more pronounced as the size of the Table 11.8 Relative growth es of plasmid-ieeand plasmid caryingcals (From 7 Iman andS Aiba, 1981, A perpen he ‘tplcaton of gneienginerng bly ofrcombinent laid Ao NX. Aca, S369. 1-10) Organon Plasmid ae Eacherchia coli C600 Flee 099-110 Evcoli KIZECIOS5 Ried 19 103-112 E coli KI21R713._TP120 (vations) 1.50231 EvcoliJC7623. Col FL 29 ColEL decvative Ta 1.15-1.54 insertion (various) ColEt deletion mutant 1.06-1.65 (various) Preudomons TOL 200 seruginese ADLFigure 1.12 _Practionofplasnid-carying cells in batch culeureafics25 generations (Prom. Imanaka and S. Aiba, 1981, A perspective on the application of genetic engineering: stability of recombinant plasmid. Ann. N.Y. Acad Sei. 369, 114) plasmid or copy number increases. Some valuce ofa froma the literate are lied in Table 11.8; pically 1.0 < a « 20. Under selection pressure ee may equal sero i the plasmid ‘encodes biosynthetic enaymc for erential nutziens, loss of plasm may result in y”=0. When cis isthe ease, Fremains close to 128 the plasmid-free population cannot reproduce. F also depends on p, the probability of plasmid low per generation, which can be as high as 0.1 if segregation ‘ceur, When mutation or random insertion or deletions ae the only cause of plasmid instability, pis usually much lower a about 10° Plasmid fragmentation within a hos eell can cca with higher frequency if the cloning vectors inherently uuneable, Batch culture of microorganisms usually requires 25 cell senerations oF more. Results for Fafier 25 generations have ‘been calulated from Eq, (11.68) and are shown in Figure 11.12 8 function of p and a. Feteioraes substantially as increas from 1.0020. Culrureswith p< 0.01 and a Late relatively table, with Fafte 25 generations remaining close to 1. Furthee application of Fg, (11.64) illustrated in Example ny. Example 11.7 Plasmid instability in batch culture — A plasmid-containing train of E.collis used vo produce recombinant protein in a 250-litue fermenter. The probability of plas- mid loss per generation is 0.005, The specific growth rate of plasmidcfrce cells is 14h”; the specific growth-rate of plasmid:earing cells is 1.2h-', Estimate the fraction of plasmid-beating cells afer 18 h growth ifthe inecalum contains ony cells with plasmid, Solution: ‘Themumber of enerations of plasmid-carryingcellsin 18 his calculated from Bq. (11.6 Gah) 18h 3k 2 Sabyitting this inca Bg, (11.64) with p = L= 1.17 = 0.005 s 9 To.005 ‘Therefor, after 18 h only 4596 of the cells comtain plasmid Alternative models for gsowth with plan instability have been developed [13]; some inchide equations for substrate ‘slsarion and produc formation [1d]. A weakest inthe sim- ple model presen here is the assimprion that all plasmid containing cells are the same. In realty thee are dif. Yaw slit ferences im copy number and therefore specific prow rate berween plasmid-carrying cells: probably of plasmid loss ko varies from cel ro cell. Mote complex models that recog rise the segregated nature of plhumid populations are available 05.16).