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From 2014.igem.org

Contents 1 Introduction 1.1 What’s Interlab-Study? 1.2 Official requirements 1.3 SYSU-China in Interlab 1.4 Design and Process outline of SYSU-China

Introduction

What’s Interlab-Study?

This year, all teams are invited to participate in the first international interlab measurement study (“interlab” or “interlab-study” for abbreviation), a subordinate but independent project in measurement track. In this part, all team are required to obtain the data from the same target with limits on biobrick devices, but there is still freedom of experiment design during the process (e.g. experimental apparatus and data processing method).

The goal this year is to measure the GFP expression from three specific devices, two of which should be constructed by ourselves. Besides, additional options are using RFP to conduct the same experiment, measuring fluorescence of single cell, and testing more devices.

Since correct promoter intensity stated in the instruction of official part website is extremely important for each team to construct proper bricks, these data gathered from each lab are beneficial to evaluate the stability of biobrick function. It can be indicated that the interlab data might help further understand the iGEM biobrick system and the gist of standardization of synthetic biology.

Official requirements

Measure GFP in:

1. Existing device: BBa_I20260 (J23101-B0032-E0040-B0015) in the pSB3K3 vector.

2. New device to be built by the iGEM team: BBa_J23101 + BBa_E0240 (B0032-E0040-B0015), must be built in the pSB1C3 backbone.

3. New device to be built by the iGEM team: BBa_J23115 + BBa_E0240 (B0032-E0040-B0015), must be built in the pSB1C3 backbone.

Extra Credit can be obtained by:

1. Measure cell-to-cell variation for the three required devices.

2. Build and test at least three devices that are identical to devices that you have measured, except that they express RFP rather than GFP.

3. Build E0240 devices with the entire Anderson library of constitutive promoters (J23100-J23119) following standard BioBrick protocols and submit measurements for GFP expression for all devices.

SYSU-China in Interlab

->We successfully finished in constructing the two plasmid needed in requirements.

->We obtained GFP Fluorescence data by plate-reader for the three devices and additionally calculated RPU (Relative promoter unit) for better description of promoter intensity of GFP expression.

->We obtained GFP flow cytometer (FCM) data for the three devices as a single-cell reference.

->We obtained RFP data of the three devices, but due to extremely unstable result, we waive to submit RFP data.

Design and Process outline of SYSU-China

We choose microplate reader and FCM to measure the florescence data. Our experiments in this part contains following procedures (also see Fig-1):

1. Discussion and design of the experiment

2. Transformation of the biobricks into E.coli DH5α

3. Constructing the device, which includes:

Obtainment of required plasmids, restriction analysis and gel extaction, ligation of the required plasmids, cloning and colony PCR test, and sequencing of the ensured clones.

4. Data measuring, which includes:

Acquisition of the measurements by microplate reader and analysis, acquisition of the measurements by the flow cytometer and analysis, and repeating and analysis of data and refining protocol.

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For further detailed protocol please turn to page <a href="https://2014.igem.org/Team:SYSU-China/content.html#Judging/Interlabstudy/Protocol" target="_blank">Protocol</a>; for results and data analysis, please turn to page <a href="https://2014.igem.org/Team:SYSU-China/content.html#Judging/Interlabstudy/Result" target="_blank">Result</a>.