Team:USP-Brazil/InterLab

InterLab Study

Introduction

In the thirteenth iGEM edition happens the fourth Interlab Study. This study is based on the characterization of standard biological parts and, as standard parts, it is fundamental to observe reproducibility and repeatability on their behaviour. For instance, even well characterized promoters in a given strain of E. coli may behave reasonably different in another strain. Acknowledging this challenge, the Interlab Studies is a way to gather experiments from all around the world and provide a more unified understanding about the fundamental building blocks of Synthetic Biology. Since 2016, in an attempt to standardize the obtained data, specific protocols and calibration samples were provided for each iGEM team attending the Interlab. With this approach, we can construct a rich knowledge base of standard biological parts, together with several study cases of different protocols and other details. The value this have to the whole community of Synthetic Biology is beyond doubt.

We have done not only the standard plate reader, and flow cytometry assays, but also single cell analysis by fluorescence microscopy. We have also provided better measuring conditions (M9 media was preferred instead of LB, due to high levels of auto fluorescence of the latter) and alternative approaches for quantitative assays using Do-it- Yourself methods (digital camera image analysis in a range of different setups).

Thus, we have stablished a robust comparison between all test devices and fulfilled the extra credit requirements by searching for optimized measurement protocols and generating new cheaper and accessible approaches for assessing promoter strength.

We have received six Test Devices and one positive control, which differ in respect to their constitutive promoter and RBS sequences. The devices are generally composed of a constitutive promoter, an RBS, a GFP reporter gene and a terminator. All promoters were derived from the Anderson’s library, a constitutive promoter library generated by single mutations, which affected the promoters’ strength in different ways. In this set we have pairs of Test Devices sharing the same promoters, but with divergent RBS sequences and the main reason of this arrangement was to test the influence of different RBS sequences in translation efficiency while using the same promoters.

Thus, Test Devices 1 and 4 share the promoter J23101, but have different RBS (B0034 and J364100, respectively); Test Devices 2 and 5 share the promoter J23106, but have different RBS parts (B0034 and J364100, respectively) and Test Devices 3 and 6 share the promoter J23117, but have different RBS parts (B0034 and J364100, respectively). The negative control consist only on an inert sequence derived from the TetR operator. It is important to highlight that the J364100 RBS is is one of the bicistronic design elements designed by Mutalik et al. in the 2013 Nature publication. Specifically, this is bicistronic design element number two (or BCD2, for short).

All devices and controls have the pSB1C3 plasmid (high-copy number) as a backbone. Following the iGEM protocol, all plasmids were transformed into DH5α E. coli cells - following the iGEM transformation protocol - which were used as samples for all the different experiments. You can find more information about the devices below and on figure 1.

  • Positive control - BBa_I20270 (PC) - J23151.B0032 E0040.B0010.B0012 in pSB1C3
  • Negative control – Bba_R0040 (NC) - R0040 in pSB1C3
  • Test Device 1 - BBa_J364000 (TD1) - J23101.B0034.E0040.B0010.B0012 in pSB1C3
  • Test Device 2 - BBa_J364001 (TD2) - J23106.B0034.E0040. B0010.B0012 in pSB1C3
  • Test Device 3 - BBa_J364002 (TD3) - J23117.B0034.E0040. B0010.B0012 in pSB1C3
  • Test Device 4 - BBa_J364003 (TD4) - J23117.J364100.E0040. B0010.B0012 in pSB1C3
  • Test Device 5 - BBa_J364004 (TD5) - J23117. J364100.E0040. B0010.B0012 in pSB1C3
  • Test Device 6 - BBa_J364005 (TD6) - J23117. J364100.E0040. B0010.B0012 in pSB1C3

Multi-scale combined experiments:

In order to best characterize the Biobricks, we have done experiments with differential sensibility thresholds, ranging from macroscopic analysis of photos taken by a cellphone camera to single cell analysis in a flow cytometer. The main rationale was to compare methods focused on different scales of the same system, providing both general and specific information about the behavior of the selected promoters. Furthermore, we wanted to try new inexpensive methods as regular macroscopic image analysis taken by a cellphone or digital camera. We present below an overview of those multi-scale approaches chosen by our team during the InterLab, from the macro to the cellular microscopic view:

  1. Macroscopic Analysis: Digital Camera and Cellphone Photos quantitativelyanalyzed by open-source softwares
  2. Population Analysis I: Plate Reader Assay
  3. Sub-Population/Single Cell Analysis: Flow Cytometry Assay
  4. Microscopic Analysis/Single Cell: Fluorescence Microscopy

References and Protocols

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