Team:BCCS-Bristol/Project/Wet lab

From 2009.igem.org

BCCS-Bristol
iGEM 2009


BioBricks
Notebook
Lab Photos
BioScaffold
Check out our selection of submitted BioBricks! View our team's lab book, charting our progress throughout the course of the project. Take a look at our photos from the wet lab. In-frame fusion of arbitrary BioBricks with no scarring? No problem! Read about our BioScaffold solution here.


Introduction

We wanted to exploit the fact that outer membrane vesicles (OMVs) were naturally produced in gram negative bacteria such as E. coli. The aim was to use them in our advantage as a directed delivery system to cells of proteins of our interest.This can potentially be used for the safe delivery of drugs into cells or for cell-cell communication purposes.We believe that the inclusion of proteins in vesicles will not only protect them from degradation in the extracellular environment but will also protect other cells if the cargo proteins packaged into vesicles are toxic or harmful.

Because the exact mechanism by which OMVs are produced in gram negative cells is not yet elucidated (although 3 models have been proposed, Lauren M. Mashburn-Warren et al, 2006) we decided to make protein fusions of proteins that were normally included in OMVs with proteins of our interest. Experiments with protein fusions to the toxin ClyA were already made with success (Jae-Young Kim et al, 2008) and the desired proteins were delivered to OMVs. But for the purposes that we would be using OMVs we thought that it would be safer to use harmless proteins that would act as carriers instead of ClyA. Hence we selected 3 possible candidate protein carriers (Eun-Young Lee et al, 2007) to be used in our project.These proteins are: 

                                      1. OsmE (Osmotically inducible lipoprotein E)
                                      2. fiu (siderophore receptor)
                                      3. FhuA (Ferrichrome-iron receptor)


In order to assess the production of our fusion proteins and at the same time monitor their introduction into the OMVs we used GFP (Green Fluorescent Protein) as our cargo protein fused to one of the three carriers stated above.


If you want to find out more about our project, as far as the laboratory work in concerned, click on the available links!!


Safety Issues

As the idea of communication between a certain population (in this case E.coli) could raise issues in health and safety of the general public the following precautions were taken during the implementation of the project in the laboratory:


  • Novel proteins handled (FhuA,OsmE,Fiu) were derived from DNA by gene cloning with PCR from E.coli MG1655 a laboratory strain with no toxic implications.
  • Novel proteins handled where screened from the literature to ensure that they will have no toxicity effects.
  • Experiments were implemented in a Level 1 Laboratory with access only by trained individuals.
  • Students involved in experimental work in the laboratory were trained to an appropriate level to apply relevant techniques and use relevant equipment and where suitable were supervised whilst carrying out laboratory work.
  • Laboratory workers were always clothed in appropriate manner (lab coat, gloves, safety spectacles).
  • Laboratory workers sterilised their hands before and after laboratory work and before entering and exiting the lab at all times.
  • No bacterial cultures exited the laboratory unless they were suitably packaged and accompanied by one of the team members whilst in transport and this only occurred where it was necessary to transport cultures from one laboratory to another.
  • No purified DNA or biological material was left unattended at any time, and all DNA and biological material was suitably stored according to Level 1 Laboratory rules.
  • Biosafety guidelines where followed under the BCCS-Bristol iGEM'09 supervising team and such guidelines fall within the description of a project that holds approval by the iGEM supervisor Dr.Nigel Savery.


The BCCS-Bristol iGEM'09 team declares that to the best of its knowledge that none of the Biobricks created and submitted to the partsregistry database is toxic/lethal or in any way malicious to individuals, at the time of writing this (21 October 2009).


Biobricks created and entered to the partsregistry database can be found here.

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