Team:Leiden/RPM

iGEM Leiden Wiki : RPM

RPM

Random Positioning Machine

In space, many conditions are vastly different from what life is exposed to here on Earth. Though we can protect ourselves and bacteria to some of these conditions passively (e.g. through temperature and radiation shielding), changes in gravity cannot be evaded.

This led us to pose the question: “What are the effects of microgravity and Martian gravity on gene expression in E. coli , compared to gene expression under Terran gravity?” But how on Earth can we study reduced gravity… on Earth? To this end we used the random positioning machine (RPM) by Airbus Defence and Space Netherlands, which allows us to simulate partial and microgravity. ¹

We decided to take on an ambitious approach and sequence E. coli ’s entire transcriptome, using state-of-the-art RNAseq. This allows us to see every RNA inside E. coli at a given time, providing a helicopter view of the changes occurring inside the bacteria. Though microgravity has been studied many times before, we are the first to study the effects of Martian gravity in E. coli . Moreover, many previous microgravity experiments involved microarrays, which can only detect changes in RNAs which are already known. RNAseq on the other hand, sequences RNAs indiscriminately. Following our RPM experiment, RNAseq was performed by BaseClear and subsequent analyses were performed by us.

The eventual goal of this experiment is to verify that our perchlorate reduction system will be expressed under Martian gravity. Furthermore, we will search for highly expressed genes that react in a gravity dependent manner and use the accompanied regulatory sequences to develop a biobrick for gravity dependent gene expression.

Video 1: RPM demonstration

The effects of low orbit gravity
Multiple studies in the International Space Station (ISS) showed that in many cases bacteria grow faster, show increased virulence, and can even be less susceptible to antibiotics when growing in the microgravity of Lower Earth Orbit (LEO). ^2,3 We are keen to find out whether the effect of Martian gravity resembles that of microgravity, or if Mars is more reminiscent of Earth with respect to bacterial growth. Before conducting costly and time-consuming RNAseq, we characterized E. coli (BW25113) growth through visual inspection of colonies in three conditions:

Gravity on Earth: 1g
Martian gravity: 0.38g
Microgravity: 0g

Figure 1. Source: http://mars.nasa.gov/allaboutmars/facts/#?c=inspace&s;=distance

Of course, we cannot actually travel to Mars to experience its gravity, but instead, we used the Random Positioning Machine (RPM 2.0) by Airbus Defence and Space Netherlands. This machine constantly rotates the samples over three axes, averaging gravity out on longer timescales. By reprogramming the motion pattern of the RPM so that it will stay longer in the bottom part, the sample experiences (simulated) partial gravity.

Results: visual inspection (0g vs. 1g)
E. coli BW25113 was plated on LB and in duplo exposed to microgravity in the Random Positioning Machine. Just besides the RPM, plates with the same dilutions were placed at ‘normal’ surface gravity of the Earth. After less than 22 hours, the difference between the plates (in and next to the RPM) were clearly visible: colonies are larger when grown in microgravity than when grown in 1g. Two plates are shown below as an example:

Figure 2: Growth comparison under different gravitational conditions.

This reaffirms the expected change in growth and bacterial physiology under reduced gravity. Note that these bacteria were grown at room temperature for practical reasons. The subsequent RNAseq experiment was performed with the RPM enclosed in a stove at a constant 37ºC.

Results
RNAseq (0g vs. 0.38g vs. 1g) Below, you can read the abstract of the RNAseq experiment. Click any of the links to the RNAseq page for the full report.

Abstract We compared E. coli BW25113 bacteria under three different conditions: controls (1g) simulating Martian (± 0.4g) and microgravity (± 0g). Subsequently, RNA isolation and state-of-the-art RNAseq were performed, revealing significant down regulation of a translation initiation factor (yciH, p_FDR < 0.05) and increased expression of two membrane proteins (yceK, hdeD, p_FDR < 0.1). Furthermore, under simulated Martian gravity, 56 antisense RNAs were found to be significantly differentially expressed, constituting 56% of differentially expressed RNAs. Genome annotation (GO) of the genes inhibited by the affected antisense RNAs in the 0.4g treatment shows that E. coli specifically appears to suffer from carbon-deprivation and acid stress. These results are intuitive, given the reduced or absent convection in partial and microgravity, respectively, leading to build-up of waste products. We conclude that E. coli primarily responds to perceived changes in gravity on a translational level.

References