Effect of low density lipoprotein on DNA integrity of freezing-thawing boar sperm by neutral comet assay

A modified protocol of neutral comet assay was utilized to assess the effect of low density lipoprotein (LDL) on the DNA integrity of boar freezing-thawing semen. The results demonstrated that the method was high sensitive and easier manipulation and LDL significantly protected sperm DNA integrity (p<0.05) from the damage caused by cryopreservation except TD at the concentration of 6%, 7% and TM at 6%, the optimal LDL concentration in diluents was 9%. Moreover, LDL showed better protection in 0.25 ml than in 0.5 ml types of straw (p<0.05) and no difference was observed in the same volume straw at the concentration of 9% and 10%. It was just the same for LDL effect on boar sperm DNA in cryopreservation 0 day and 30 days (p>0.05).     

Efficient strategies to enhance plasmid stability for fermentation of recombinant Escherichia coli harboring tyrosine phenol lyase

Objective

To solve the bottleneck of plasmid instability during microbial fermentation of l-DOPA with recombinant Escherichia coli expressing heterologous tyrosine phenol lyase.

Results

The tyrosine phenol lyase from Fusobacterium nucleatum was constitutively expressed in E. coli and a fed-batch fermentation process with temperature down-shift cultivation was performed. Efficient strategies including replacing the original ampicillin resistance gene, as well as inserting cer site that is active for resolving plasmid multimers were applied. As a result, the plasmid stability was increased. The co-use of cer site on plasmid and kanamycin in culture medium resulted in proportion of plasmid containing cells maintained at 100% after fermentation for 35 h. The specific activity of tyrosine phenol lyase reached 1493 U/g dcw, while the volumetric activity increased from 2943 to 14,408 U/L for l-DOPA biosynthesis.

Conclusions

The established strategies for plasmid stability is not only promoted the applicability of the recombinant cells for l-DOPA production, but also provides important guidance for industrial fermentation with improved microbial productivity.

     

Enhanced Expression of Adenovirus Encoding rhEPO Assisted by BAPTA

Infection efficiency is the key issue for gene delivery using adenovirus vector and usually unsatisfactory. In this study, recombinant adenoviruses encoding recombinant human EPO were prepared using the Adeasy system, and injected into the mammary gland of goats via the teat canal. BAPTA was used to treat the mammary gland to facilitate adenoviruses infection compared with EGTA. Milk serum was collected from the infected mammary gland and characterized by ELISAs and Western blotting. Expression level of rhEPO from the group treated by BAPTA was higher than that treated by EGTA.