Team:Calgary/InterLab

Header

The InterLab Study

Introduction

The InterLab study is an international collaborative lab study where labs around the world perform identical procedures to establish reliable and repeatable measurements and protocols in synthetic biology. This year, the InterLab study focused on GFP expression in E. coli DH5ɑ using different ribosome binding sites and promoters. GFP was measured via fluorescence readings on a plate reader, and a standard measurement procedures were given out by iGEM to all participating teams. This universal protocol is critical, as it enables data to be compared with as many constant variables as possible to ensure that comparisons are accurate.

This study involved two different ribosome binding sites tested with three different promoter regions, along with a positive and negative control, for a total of 8 different trials. Each trial was compared based on its GFP fluorescence readings. The procedure that we followed for this study, which was distributed by iGEM, can be found here.

Provided below are the plasmid constructs of each trial, as well as a brief summary of what each part does. All parts were received from the iGEM registry and can be studied more thoroughly on the iGEM site by clicking on their names below. Also provided are graphs showing the absorbance and fluorescence readings from our InterLab experiment.


Parts & Plasmids

BBa_E0040: This part is common across all of the transformed plasmids in the 2017 InterLab study (except for the negative control) and codes for the production of GFP. GFP, or green fluorescent protein, is a protein native to the Aequeora victoria jellyfish, which glows green when exposed to UV light. GFP is used here as it provides a visual result that can be measured to compare expression of the GFP protein amongst the various plasmids.

BBa_0010 and BBa_0012: Another couple of parts that are common to all of the InterLab plasmids; BBa_0010 and BBa_0012 are both transcriptional terminators that stop RNA polymerase from transcribing DNA into RNA. Simply put, these parts end the genetic circuit.

Chloramphenicol resistance: This part gives resistance to the antibiotic chloramphenicol, allowing bacteria containing this plasmid to be grown on chloramphenicol-containing agar plates. Chloramphenicol resistance is built into the pSB1C3 backbone.

BBa_B0032: This part codes for a ribosome binding site, the location on RNA where ribosomes bind to begin protein translation. BBa_B0032 is considered a weak binding site when compared to BBa_B0034, and is only used in the positive control.

BBa_B0034: This part codes for a ribosome binding site, the location where ribosomes bind to RNA to begin protein translation. BBa_B0034 is one of the two ribosome binding sites tested in this experiment.

BBa_J364100: This part codes for a ribosome binding site, the location where ribosomes bind to RNA to begin protein translation. BBa_J364100 is one of the two ribosome binding sites tested in this experiment.

BBa_J23151: This part is a promoter region, which recruits RNA polymerase to begin transcription of DNA to RNA. BBa_J23151 is only used in the positive control.

BBa_R0040: This part is a promoter region, which recruits RNA polymerase to begin transcription of DNA to RNA. BBa_R0040 is repressed in the presence of tetracycline, and is only used in the negative control, not in any of the actual tests.

BBa_J23101: This part is a promoter region, which recruits RNA polymerase to begin transcription of DNA to RNA. BBa_J23101 is one of the three promoters tested in this experiment.

BBa_J23106: This part is a promoter region, which recruits RNA polymerase to begin transcription of DNA to RNA. BBa_J23106 is one of the three promoters tested in this experiment.

BBa_J23117: This part is a promoter region, which recruits RNA polymerase to begin transcription of DNA to RNA. BBa_J23117 is one of the three promoters tested in this experiment.


Graphs of Results

Figure 1: The absorbance graph shows the amount of light absorbed by the sample at a wavelength of 600 nm, which is a direct reflection of the cell density in the sample. This effectively shows the amount of bacteria within each trial.
Figure 2: The fluorescence graph shows the amount of light emitted by the samples, and represents the amount of green fluorescent protein within each trial.
Figure 3: The fluorescence/absorbance graph is the most accurate graph for measuring protein expression in this experiment, as it accounts for amount of GFP and number of cells present. This way, data are not skewed if more or fewer cells are present in one trial. Using this data, Test Device 1 (BBa_J23101 and BBa_B0034) tended to have the most effective GFP expression.


Procedures provided by iGEM were strictly followed, and all InterLab data and completed forms were submitted to iGEM HQ.