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From 2012.igem.org

Contents 1 States 1.1 Containment of Bacillus subtilis spores: the sticker 1.2 Germination 1.3 Activation Pathways 1.3.1 First strategy: TnrA pathway 1.3.2 Second strategy: identification of “PBADmeat” 1.4 Color reporter 1.5 Death

States

Containment of Bacillus subtilis spores: the sticker

The spores of Bacillus subtilis will be contained in a compartment which is impermeable to bacteria and liquid, but can still let through volatiles. Metabolites, water, and alanine should be separated from the spores by a breakable membrane. Mixing these compounds will cause germination.

The sticker itself is made of a materials that is able to let go gases inside but keeps liquids, spores and bacteria inside and sealed off from the environment, see our safety page. For this purpose TPX polymer would suit the best because it has pores with a size of 0.5um. We want to make a prototype for testing our Bacillus spores.

Germination

Alanine and water can trigger germination of Bacillus subtilis. The modeling team will provide information on the optimal concentrations for germination, and on the germination time.

Activation Pathways

We follow two different strategies to build a pathway in Bacillus subtilis which can react to the presence of rotten meat volatiles. One is using the TnrA pathway, which is normally expressed in absence of ammonium (reference). For the other strategy, we use a transcriptomics approach searching for promoters present in Bacillus reacting to rotten meat volatiles.

First strategy: TnrA pathway

Pre-Activation

1. The growth medium contains a low concentration of glutamine (gln), but no ammonia or ammonium (NH4).
2. Glutamine synthetase (GS) is activated due to the inadequate level of glutamine, however, it cannot synthesize more gln as it is lacking NH4.
3. The low intracellular gln level shifts the ratio of GS to feedback-inhibited GS (FBI-GS) towards a higher level of GS.
4. This higher level of GS allows a higher level of TnrA.
5. The TnrA represses the alsT promoter.

Activation

1. Rotting meat produces NH4.
2. NH4 is enters the cell through the NrgAB ammonium transporter.
3. GS converts NH4 into gln.
4. Gln reaches the concentration required for steady cell growth.
5. The ratio of GS to FBI-GS shifts towards a higher level of FBI-GS.
6. The newly created FBI-GS binds to TnrA, creating an inactive complex.
7. TnrA is unable to repress the alsT promoter.
8. No longer repressed, alsT activates along with its positive feedback loop containing the color reporter.

Second strategy: identification of “PBADmeat”

TnrA is a repressor/activator which acts on a broad level. This makes it a tricky candidate for triggering a single reaction. Therefore, an alternative strategy is needed.

To identify our rotten meat volatile reporter (short: PBADmeat), we performed an transcriptome analysis of Bacillus subtilis subjected to rotten meat (target) and to fresh meat (control) volatiles. Analysis of the differential expression between these two situations can lead to candidate promoters for our construct.

For more information on the technique and some preliminary results, see the Wetwork page.

Color reporter

As a reporter, we chose to use a pigment. The big advantage of using a pigment over another type of reporter (for instance, GFP or an electrical signal) is that it can be detected without any equipment. A big drawback however, is the production speed of pigment.

To make Bacillus subtilis produce more pigment, we plan to build in a positive feedback loop. Different feedback loops are available in the Registry of Standard Biological Parts (for instance PLux/LuxR and pRE/CII). We plan to characterize and - if necessary - improve one for our construct.

We will start with using the E.chromi pigments from the iGEM team of Cambrige, 2008. These pigments (violacein and lycopene) are produced strongly in E. coli and are visible by eye at even low optical densities. To test if our construct principally works, however, we will use GFP.

As an alternative for the pigments, we we're provided with chromoproteins from Uppsala 2011. These biobricks were send to us in agar stabs and produce yellow or blue color. We might be able to test if they work in Bacillus.

Death

To make sure no live GMO will get into the environment, the death of Bacillus should be reached:

1. The extracellular nutrients are depleted.
2. Color reporter builds to toxic levels.