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SBNet - Research Reports 1998

Petra Franzén/Eva Davey

Name: Petra Franzén

Position: Laboratory Engineer, Expression Laboratory

Name: Eva Davey

Position: Laboratory Assistant, Expression Laboratory

During 1998 the number of users of the Expression lab has increased. Some people just use our equipment while others have been working in the lab for longer periods. In November Dr Diane Lester started as a post-doc for one year in Prof. Inger Andersson group. She is a molecular biologist and she will be working most of her time in the Expression lab with expression and functional studies of gibbevellin 3-ß-hydroxylase.

Parts of this year have been spent in investing in new equipment and to organise and establish working routines for the lab. The rest of the time I have spent helping and advising people in their various projects.

Rebuilding and equipment

A small part of our lab has been rebuilt to be able to have a separate room for all work with bacteria. In this room there are two shakers, a 5 L fermenter, a small spectrophotometer and a sonicator. The RC-5 and RC-3 Sorvall centrifuges and their accessories have been repaired and upgraded. In the beginning of 1999 we will install a new centrifuge, RC-26, that is a version of the RC-5 centrifuge with a stronger motor. A new lightweight rotor for the RC-5 and RC-26 (6x500 ml) has also been purchased. Two important new investments are an advanced spectrophotometer from Beckman and a Robocycler from Stratagene. With the spectrophotometer it is possible to perform simple fixed wavelength analysis as well as wavelength scanning and time-based kinetic determinations. With the ultra microcuvette it is possible to perform measurements in as small volumes as 2-5 ul. Along with the spectrophotometer a small water bath has been bought to make it possible to regulate the temperature in the cuvette during the measurement which is important during kinetic studies. The Robo-cycler is a PCR machine with a temperature gradient function over the annealing block. The robot function makes it much faster than the standard PCR machine and by using the gradient function it is possible to determine the optimal annealing temperature without performing several runs.

Working routines

Another role of the Expression lab is to centralise expensive material e.g. restriction enzymes and other enzymes, plasmids, bacterial strains, molecular biology kits etc. Time has been spent to organise convenient systems and protocols for these things. To further simplify for the lab working guests, there are two fully equipped lab benches and the guests will be provided with what they need to get started e.g. pipettes, sterile tips, sterile tubes, competent bacteria, medium and antibiotics for small culture volumes. Information regarding the Expression lab and its equipment has been presented to the structural biology groups in Uppsala (TAJ/SM, Eklund, Liljas/Unge and Hajdu/Andersson/Knight). Here the importance of communication, and the fact that students or post-docs without experience in the field of molecular biology and recombinant protein expression must have a supervisor, who can help to design, perform and interpret the project, was stressed. It has also been "guided tours" in the lab.

Future

During 1999 BMC will be reorganised. The Expression lab will stay in its present location and may be able to get some more space when "our corridor" D11.2 will be rebuilt partly. Our aim is to expand the equipment so that it will be possible to express various kinds of proteins, not only the "easy ones". Some mammalian proteins have been shown to be inactive or insoluble when expressed in bacterial systems. To overcome these problems one can either try to refold the insoluble protein or use another expression system. Therefore, we want to introduce eukaryotic expression system(s), i.e baculo expression system. In order to set up such systems it is necessary to build a separate cell culture room for sterile work. There are also plans for facilities and equipment that will make it possible to perform various kinds of functional studies of the recombinantly expressed proteins. This is what I will be focused on in the nearest future provided that the Expression lab will get access to more space.


SBNet Latest update at 18 February, 1999.