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From 2010.igem.org

Contents 1 Salt Tolerance 1.1 Aim 1.2 Proposed Method 1.2.1 Step 1: BioBrick Formation 1.2.2 Step 2: Salt Tolerance Characterization

Salt Tolerance

Aim

To create BioBricks that will facilitate an increased halotolerance against NaCl in the host organism (E.coli). These BioBricks will be implemented in Escherichia coli K12, characterized and evaluated on halotolerance as well as any influential effects on protein expression.

Proposed Method

This sub-project will focus on creating an Escherichia coli K12 strain which is capable of withstand the high salt (NaCl) concentrations present in the world’s oceans. This will be done by implementing the bbc1 gene (from Chlamydomonas sp. W-80) in E.coli, using the “BioBrick” method. Once this has been achieved the effects of bbc1 on the halotolerance of the strain will be characterized as well as the effects of bbc1 on the production of other proteins (using GFP).

Bbc1 is an Algal protein, which has a high number of homologues in multiple species. Although the exact function is not know it has been shown to have an influence on the increase the halotolerance of Chlamydomonas sp. W-80. It has been successfully expressed in E.coli, and was shown to increase the salt-tolerance significantly (Tanaka et al. 2001). Based on: Satoshi Tanaka, Kazunori Ikeda, Hitoshi Miyasaka. Enhanced Tolerance Against Salt-Stress and Freezing-Stress of Escherichia coli Cells Expressing Algal bbc1 Gene. Current Microbiology. 42:173–177(2001)

Step 1: BioBrick Formation

The necessary gene, bbc1, will be synthesized, and the appropriate RBS and promoter that will be used are existing BioBricks. These components will be combined with a pSB3T5 plasmid. Three plasmids will be made, one containing the BBa_J23119 promoter, one containing the BBa_J23116 and one containing BBa_J23107. Where BBa_J23119 has a strong transcription level and BBa_J23107 has a (relatively) medium transcription level and BBa_J23116 has a low transcription level. This will allow us to detect the effects of differing concentrations of bbc1 on salt tolerance as well as the production of other proteins. The transformed cells will be cultivated in shake-flasks of 250ml. The final constructs (3) will contain the bbc1 gene under a high transcription level promoter (construct 1, BBa_J23119) as well as a low(er) transcription level promoter (construct 2, BBa_J23107 and construct 3, BBa_J23116), and the necessary RBS on (medium copy) pSB3T5 plasmids.

Step 2: Salt Tolerance Characterization

Strains:

• bbc1 (high transcription): E.coli K12/100T
• bbc1 (medium transcription): E.coli K12/101T
• bbc1 (low transcription): E.coli K12/102T
• Negative control: E.coli K12

In order to characterize the BioBricks, the strains containing one of the BioBricks will be cultivated under different salt concentrations. Of each strain, 4 cultures of 100ml will be characterized, each containing different salt concentrations, ranging from 0M up to 0.8M NaCl. OD600 measurements will be done in order to determine the growth curve and SDS PAGE will be used to show whether the protein is being produced.