Overview

How do standard promoters behave in Mountain View, California, USA? We participated in the 2nd Annual InterLab Measurement study to contribute our results to a body of data from teams from around the world!

See our BioBricks

Background

The purpose of the 2015 InterLab Study is to measure the fluorescence of three devices: a high-, medium-, and low-strength promoters fused to a green fluorescent protein (GFP) generator. iGEM teams from around the world construct the same devices and compare results.

The three devices are:

Device 1: J23101 + I13504, in pSB1C3

Device 2: J23106 + I13504, in pSB1C3

Device 3: J23117 + I13504, in pSB1C3

For our positive control, we used the one suggested: GFP device BBa_I20270. For our negative control, we also used the one suggested: TetR repressible promoter.

All devices and parts were obtained from the 2015 Distribution Kit. Please see the link to our lab notebook below for more detailed information on device assembly. The results were confirmed with DNA sequencing by Elim Bio.

We also participated in the Extra Credit Opportunity.

Data and Results

There were two experiments, recreating HYDRAs from the Chen et al. publication, and creating bioHYDRAS, which are fully biological versions of HYDRAs.

Recreating HYDRAs: By expanding on the protocol by Chen et al. (1) to create HYDRAs, we then used desiccant and wet paper towels in separate chambers to create humidity variance for these HYDRAs to expand and contract, and recorded our results using a ruler and a humidity sensor.

BioHYDRAs: The goal of BioHYDRA was to replace all the parts of HYDRAs by biologically produced substances. We sought out to replace polyamide tape by bacterially cellulose, and the glue by cellulose binding domains on the surface of the spore coat. Thus, the first step involved cloning a Bacillus construct in Escherichia coli of a fusion protein sequencing consisting of a spore coat protein, cotZ (building off work done on Sporobeads by the LMU Munich 2012 iGEM team), and a cellulose binding domain (CIPA). Additionally, we decided to add aeBlue, a chromogenic protein, between cotZ and CIPA to be able to see with the naked eye whether Bacillus is in a vegetative or a spore state. The plasmid would thereafter need to be transformed and expressed in Bacillus . We then needed to produce bacterial cellulose. For more details, refer to our Cellulose page. Finally, our project would consist of testing for the binding affinity of the spores on the cellulose before we could construct our bioHYDRAs. To do so, we used the cellulose binding affinity protocol that the 2015 Edinburgh team sent us in light of our collaboration.

Protocols

The chassis we used for the InterLab Study was E. coli NEB5-alpha, which is Biosafety Level 1. The personal protective equipment used included gloves and long pants. We used BioBricks assembly to construct all of the devices, and verified the sequences with sequencing done by Elim Bio.

To grow up the cells, we completed the following steps as specified in the InterLab Study Protocol Form:

1. Streak out one plate of LB agar with appropriate antibiotic per device and control.
2. Incubate plates for 17 hours at 37C (New Brunswick INNOVA 4200 Shaker Incubator).
3. Inoculate three biological replicates of 4mL liquid cultures in LB + chloramphenicol 15mL conical tubes.
4. Incubate liquid cultures for 18 hours at 37C, shaking at 190RMP and placed at an angle.
5. Samples were placed in 4C fridge for 24 hours before testing.
6. Samples were diluted 1:20 in LB and measured on a flow cytometer.
7. Use a plate reader and 96-well plate to perform OD600 and fluorescence measurements, using 200uL of samples and LB + chloramphenicol blanks.
8. Set instrument to read OD600, take measurements, calculate the dilution required to reach an OD600 of .5 ± 5%, perform the dilutions, and re-measure. Repeat this step until the OD600s are all .5 ± 5%.
9. Place 96-well plate with samples into fluorometer and measure with excitation at 485nm, emission 528nm, cut-off 495nm.
10. Plate reader data was processed using Microsoft Excel.