Project Overview

This Project aims to solve the cooling problem that has been present since the beginning of time. With our BactoCooler, you will not have to worry about Global Warming anymore however there is the possibility of a future Ice Age. Joke aside, we as humanity have no clue about how much potential bacteria carry in cooling down the environment. For the cooling process with bacteria, we needed to designate an organic and endothermic reaction to use. As the reaction, we chose the breakdown of urea. Because we chose urea as the target of the breakdown, we were required to implement the urease enzyme.

There are certain problems that arise as a result of the constant cooling, both for the bacteria and the environment. A process as efficient as this one had to be under control. For this reason, we had to put up a control mechanism in our BactoCooler. The solution was the RNA Thermometer…

For any protein to be synthesized, the ribosome must first attach to the RBS of the mRNA. RNA Thermometers are also Ribosome Binding Sites. The differentiation between RNA Thermometers and the other RBS is the functioning of the Shine-Dalgarno sequence.The Shine-Dalgarno sequenceis a ribosomal binding site in the mRNA, generally located 8 bases upstream of the start codon AUG. The Shine-Dalgarno sequence exists both in bacteria and archaea, being also present in some chloroplastic and mitochondial transcripts. The six-base consensus sequence is AGGAGG; in E. coli, for example, the sequence is AGGAGGU. This sequence helps recruit the ribosome to the mRNA to initiate protein synthesis by aligning it with the start codon. This sequence is sensitive to the changes in temperature in RNA Thermometers. The Shine-Dalgarno sequence has enclosed structure under certain temperatures and this results in the preventation of translation. With increasing temperatures, the conformation of the sequence leisurely changes to increase the rate of translation until the optimum temperature where the rate of translation is at it’s highest.

After the control mechanism, the RNA Thermometer, comes the sequence of the urease enzyme part which is the main actor of the endothermic reaction desired. We initially wanted to directly control one urease enzyme through adding the part after the RNA Thermometer part; the fact that the fully functional breakdown of urea requires 3 seperate enzymes at the same time was a downfall on our behalf. The initial subunit of the composite part has the biggest base length, a base length bigger than the total of the two other subunits.

For the urease enzyme to be produced as a result of translation, each subunit must be seperately synthesized causing these polypeptides to bind with each other. In our model, there is aRNA Thermometer which is followed up the long primary subunit, which is continued with a RBS and insulator plus the second subunit and the composite part is finished of with another RBS and insulator plus the third subunit. The total base length of the sequence is 2570 base pairs.

Our urease part is designed to serve one big purpose. With our system, our E. Coli won’t have to synthesize the long composite part continously. The RNA Thermometer is associated with the primary long subunit and the it’s presence will stop the production of the long and inconvinient to synthesize subunit. If the RNA Thermometer is restrictive in the presence of insufficient temperature, then the first and long subunit won’t be produced and only the second and third subunits will be synthesized. However when the RNA Thermometer is active, the first subunit will also be produced and the composite part will be crafted. Via this modelling, we have relieved our bacteria from continously producing long polypeptides. If our bacteria were to produce the primary subunit as well, it would have problems in continuing it’s life cycles.

The urease composite part will break down the urea to ammonia and carbon dioxide.

This endothermic reaction is: CO(NH2)2 + H2O →CO2 + 2NH3

In this reaction, for the breakdown of the covalent bonds between carbon in the center of the urea and the nitrites and also the covalent bonds in water, heat must be taken. When the reaction enthalpies are calculated, it can be observed that the total energy needed to break the bonds surpass the amount of energy released when forming the bonds of CO2 and NH3.

ΔHf [CO(NH2)2] = -333.51 kJ/mol

ΔHf [H2O(g)] = -285.83 kJ/mol

ΔHf [CO2(g)] = -393.51 kJ/mol

ΔHf [NH3(g)] = -46.11 kJ/mol

ΔHrxn = [2 x ΔHf (NH3(g)) + ΔHf (CO2(g)) ] – [ ΔHf (H2O(g)) + ΔHf (CO(NH2)2) ]

= +133.61 kJ/mol (The reaction is endothermic as the reaction absorbs heat from the environment.)

Teoritically, the bacteria will effectively cooldown the environment but the ammonia produced as a byproduct of the reaction puts the bacteria in mortality risk. This happens because the toxicity of ammonia is 100,000 more compared to urea. Yet, for the ammonia to be produced, there must already be urease synthesized in the environment. Even if the bacteria die as a result of the toxic environment, the reusability of enzymes will enable the breakdown of urea to occur without stopping.