Metabolic Engineering of Acinetobacter baylyiADP1 for Protein Production

Bushby, Jessica (2022) Metabolic Engineering of Acinetobacter baylyiADP1 for Protein Production. MRes thesis, University of Lincoln.

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Abstract

Vaccinations are a crucial preventative measure to stop the spread of a disease. However, due to the need to follow a cold chain procedure, the cost of transport for keeping the vaccine at +2 °C to +8 °C temperature at all times to avoid any loss of efficiency can become a hindrance in supplying vaccines. In an effort to be able to transport these essential vaccines, a new transport method is needed. By creating a new system for transport, it is hoped vaccines can be more readily available to everyone.

This aim of this research was to develop a new production system to produce an additive for modifying the formulation of proteinaceous drugs to enable thermostability. This system would increase the thermostable range of vaccinations. The protein Mannosylglycerate, MG, from Rhodothermus marinus, has the potential to work in this type of system. By isolating the mgs gene which produces this compound, a genetically modified Acinetobacter bayliiADP1strain can be produced. Using this gene and the metabolic pathway of metZ in ADP1, a microbial factory capable of biosynthesising MG can be developed. The protein could then be purified and added to a vaccination mixture and make it more thermostable in hotter temperatures.

Several different methods were used throughout the research, mainly bacterial cell culture and plasmid manipulation. Batch culture was used to observe the growth of a plasmid-containing bacterial cell culture at high and optimum temperatures, one with the mgs gene present and one without, to allow the thermostable properties of MG to be seen. This experiment produced inconclusive results as the organism containing the plasmid with the mgs gene did not grow as expected.

Following this, a shuttle vector of PCR Blunt and metZ was constructed using restriction enzyme BglII and ligation methods. This construct was planned as a base in which further plasmid manipulation was to be performed to insert the mgs gene. However, when the kanamycin resistance cassette was ligated into this shuttle vector with BamHI and BglII restriction enzymes, a crossover event occurred, and the shuttle vector had to be changed to pUC19.

A new plasmid construct of pUC19 and metZ was created using the restriction enzyme EcoRI and ligation methods, and blue/white screening was used to isolate the plasmids containing the correct insert after ligation. This new vector showed promise but due to time constraints the research was not completed. However, this did highlight the potential for using the metabolic pathway of metZ in Acinetobacter baylyi ADP1 to insert the mgs gene for the production of MG.

Keywords:metabolic engineering, protein production, acinetovacter baylyiADP1, vaccine transportation
Subjects:B Subjects allied to Medicine > B230 Pharmacy
Divisions:College of Science > School of Pharmacy
ID Code:50541
Deposited On:25 Aug 2022 10:05

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