Introduction

‘Hello World!’ are the first words a programmer prints when learning a new programming language. In analogy to this our team decided to do a ‘Hello World’ project in order to familiarize ourselves with lab techniques that we used later on to construct plasmids. Specifically we were performing PCR with uracil-containing primers, purifying PCR products and ligating them by means of USER cloning.

Since we are working with many periplasmic proteins, we wanted to try to target proteins to the periplasm. To do this, we used periplasmic signal peptides from the TAT and Sec pathways, and with a translational fusion of the signal peptide to GFP, we expressed GFP in the periplasm. Simultaneously, we expressed RFP in the cytoplasm.

Results

This image shows the bioreactor set up to run in triplicate.

Transformed E. coli taken with a confocal microscope

Close up of several cells, showing GFP expression in the periplasm

Fluorescence spectrum cross section of one transformed E. coli cell

Here is a detailed guide on how to direct GFP to the periplasm (link).

Conclusions

Biobrick BBa_K1067009 successfully directs proteins to the periplasm in E. coli.