The goal of the interlab study is to obtain GFP fluorescence data from the iGEM teams all around the world for about twenty constructions. This will permit to observe the result repeatability across technics, strains and teams.

There were two sub-parts in this study:

• The mandatory one: obtain fluorescence data for three specific genetic devices expressing GFP and compare them.
• The Extra Credit assignments: we choose to study the entire Anderson library of constitutive prokaryotic promoters (J23100 to J23119).
  This library corresponds to 19 constitutive promoters containing some nucleotide mutations on the -35 and the -10, as shown Used Biobricks and plasmids . The idea is to compare the expression strength of promoters according to mutations, from the maximum amount of fluorescence data in order to have significant results.

The three required constructions shown various fluorescence intensity at OD 600 nm = 0.5, as shown on graph and bar chart below. As shown on corrected GFP fluorescence intensity according to OD 600 nm, the relationship between corrected GFP fluorescence and OD 600 nm is linear that corresponded to the expected profile for a constitutive promoter.

Theoritically constructions I20260 and J3101 were similar, constructions and alignments in sillico matched perfectly. The difference between these constructions lies on the assembly way. However we obtained, as some other teams (University of Oxford, Paris-Bettencourt and ITESM-CEM), significant GFP expression difference. There is probably a Biobrick standard construction problem, which relates to the traited question on our Phylosophy subsection.

Our team achieved the 19 constructions of the Anderson library. Constructions were first designed in a software(Geneious v. 6.1.6)and then relized on web lab using iGEM 2014 Distribution kit. Verifications were made by eletrophoresis gel analytic PCR amplification and sequencing. Glycerol stock of 3 colonies per constructions were conserved and used for the measurments of GFP expression.
Unfortunatly, we did not collect satisfying data for the 19 constructions,although assays were repeated several times. The data profile was repeatable under people, apparatus and strain. Strain carrying promotorless vector seemed to had higher fluorescence intensity than strain containing constructions from the Anderson library, that explains negative curve on Corrected GFP fluorescence intensity according to OD 600 nm graph. However exploitable data were collected for 5 constructions (100, 104, 105, 107 and 118) for which the relationship between corrected GFP fluorescence and OD 600 nm was linear that corresponded to the expected profile for a constitutive promoter (See graph Corrected GFP fluorescence intensity according to OD 600 nm). Corrected GFP fluorescence intensity at OD 600 nm = 0.45 of these 5 constructions were presented on bar chart above and compared with registry data on table below.

Ratio were calculated dividing constructions corrected GFP fluorescence at 0.45 OD 600 nm by construction 100 corrected GFP fluorescence at 0.45 OD 600 nm.

Results were similar to registry results for the 100, 105 and 107 constructions contrary to constructions 104 and 118 for which data were significantly divergent.

Possible approach to overcome data acquisition problems:

• Using M9 medium instead of LB medium to minimize the basal medium fluorescence
• Using another E. coli strain that has a smaller natural fluorescence
• Changing of dilution and gain to find the optimal one
• Choosing a TECAN with a lower detection limits to collect data for weak promoters