Team:Leiden/Description

iGEM Leiden Wiki : Overview

RFP in Microgravity

RFP IN MICROGRAVITY

Improvement of the characterization of the RFP-construct in pSB1C3, pSB1A3 and pSB1K3
Using the Random Positioning Machine, we wanted to study whether the standard RPF construct in the iGEM backbones is functional in microgravity. This RFP construct is used for red-white screening after 3A assembly, and can besides be used for many different purposes in microbiology, for example protein localization. When using these techniques in space, as we will for sure when colonizing neighboring planets or performing experiments during space travel or on board of the ISS, it is very important to know in advance whether a testing method could be used – and our team showed testing with RFP in space is possible!

This characterization improvement shows that BBa_J04450 is expressed functionally in the three tested iGEM backbones, with resistance against chloramphenicol, ampicillin and kanamycin. To this extent, E. coli DH5α cultures were transformed with a backbone containing the standard backbone with the standard RFP Coding Device insert. The strains were grown in liquid and then plated in different dilutions. LB plates with the corresponding antibiotics were put at 37 degrees Celcius on microgravity in the Random Positioning Machine and 1g next to the machine. As a control, E. coli DH5α without a plasmid was grown, resulting in white colonies while all other backbones provided the colonies with a red to pink colony.

Pictures were made of all plates using a scanner and analyzed using FIJI (Fiji Is Just ImageJ): per plate the middle of 50 colonies (if this amount was present) was selected and the RGB values were determined. White colonies also show a high red-channel value, together with a high green and blue value. To solely determine the redness of the colony, the red-value was therefore corrected by dividing it by the sum of the red, green and blue value. This collection of values per colony was then compared between the microgravity plates grown on the RPM and the plates grown under the gravity of the Earth.

A boxplot of all redness values per colony per condition is shown below to illustrate the differences in redness found. Analysis was however performed using median relative redness per plate. By statistical analysis it was shown that no significant difference in redness and thus overall RFP expression is found between each dilution grown under microgravity (0g) or the gravity of the Earth (1g). The statistical tests are elaborated in this file . The redness values from all strains containing a RFP coding Device backbone do differ significantly from the white E. coli DH5α control, with a p-value < 0.001.

References:

  1. Melnyk, R. A., Clark, I. C., Liao, A., & Coates, J. D. (2014). Transposon and deletion mutagenesis of genes involved in perchlorate reduction in Azospira suillum PS. MBio, 5(1), e00769-13
  2. Bender, K. S., Shang, C., Chakraborty, R., Belchik, S. M., Coates, J. D., & Achenbach, L. A. (2005). Identification, characterization, and classification of genes encoding perchlorate reductase. Journal of Bacteriology, 187(15), 5090-5096