| Cyanobacteria, or cyanophyte are photoautotrophic organisms with many special properties, making them very attractive for molecular biological studies.Funded by National High Science & Technical Program (863), our lab had succed in expressing human thymosinofi (Tai) gene in cyanobacteria Synechococcus sp.PCC7942 with the suage of shutter vector system and reconstructed plasmid vector system.This experiment began with the synthetic medium BG-11 and studied various factors, eg. temperature, Light, carbon sources, nitrogen sources & pH, etc, which influence the expression Ta?i, of in transformant 7942. Based on the results from single factor research, orthogonal experimant and central composite design(CCD) are used to optimize the medium component, calture condition and culture farm A mini-auto-closed-photolioreactor was adopted for culture.The single factor research shows, using logarithatie phase cells as inoculum, 6% (v/v) for transformant and 5% (v/v) for prototrophs, the lenghth of lag period will the be shortened. The optimized temperature is 28癈~32癈, initial pH value as 9. For both strains, lighting through the culture time will increase biomass. Optimist light strenghth is ISOOLx & 2000Lx each; NaHCOs is more geneficial than Na2CC>3, range from 10 to 30mg/L and 20 to 40mg/L each; KNCh is an essential nitrogen source, and it's optimist scale is 0.7~1.7g/& 1.2-2.2g/L each.The orthogonal experiment shows, the optimist medium and the culture condition for the transformant is: NaHCOs, 30mg/L KNOs, 1.7g/L; initial pH value, 9.0, whilst 40mg/L, 2.2g/L & 9.0 for the prototrophs respectively. These factors give different influences on both strains growth: NaHCC>3>pH> KNC>3. The transformant tets a maxium lionass (ODy3o) of 1.461, specific growth rate (U ) of 0.375d"', it's 106% more than former y valae (0.182d"!). Whilst the prototroph get a maxium biomass of 1.559, n value of 0.382d"', 99% more than former u value (0.192d~').inThe CCD research shows that optimige medium and cultare condition for the transformant are NaHCO3, 36mg/L; KNO3, 1.3g/L; initial pH value 10.0; light strenghth, 1800 Lx. Under the condition mentioned above, the maxium biomass OD730 is 1.820, which gets a dry biomass of 1.08g/L and then value is 0.500d-1, 174.7% higher than initial value and 33% higher than that of orthogonal experiment. By with differential analyses biomass products in the Autophotobioreactor computer we got a fomula as below:Y=68.67 - 2.085X1 - 75X2 - 3.083× 10-2X3 - 6.632X4 + 2.21X,X2 + 6.276 × 10'4X1X3 + 0.2183X1X4 + 3.757 × 10'2X2X3 + 7.973X2X4 + 3.341 × 10-3X3X4 - 8.623×10-4X1X2X3 - 0.2252X1X2X4 - 5.717 × 10-5X,X3X4 -3.794× 10-3X2X3X4 +8.498 × 10-5X1X2X3X4-1.638 × 10-3X12- 1.086X22-1.059×10-6X32-7.475×10-2X42While Y-biomass (OD730), X1-concentration of carbon source(mg/L), X2-concentration of nitrogen source (g/L), X3-light strenghth (Lx), X4 -initial pH value, P0-control, P1-etc-the coefficients for the terms. The dicision coefficient R2 is 0.88, showing the fomula is fit for precipating cell density.And using the result from previous experiments, we culture 7942 in a redesigned photobioreactor. After the 3days culture, the maxim biomass (OD730) is 1.510, u value is 0.979d"', its 437.9% higher than flask culture (0.182d-1), 161.1% higher than that of orthogonal experiment and 95.8% higher than RSM. The result is higher than calture of cyanoacteria 7002 (0.4-0.6d~', Kang ruijuan etc.). The maxium products in this system is 0.299g/ (Ld) , higher them that of sprulinaplatensis (0.05012g/ (Ld) Liu Chuanlin etc.)Finally, the expression of UB-Ttt], is detected by SDS-PAGE and Western blot.The research work shows a high-cell density cultivation method has been reached. It will shorten the lenghth of cultare time, and derect the large-scale culture of the transformant. |
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