In modern fermentation process, cooling system, aiming at keeping the cells in a good condition, plays an important role. However, that results in a great consumption of energy. According to a research carried by COFCO, the leader of bio-industry in China, once the fermentation temperature limit can be risen by 1 centidegree, 100 million degrees of electricity can be saved all around China. Moreover, thermal electric generation accounts for a large proportion in China. Let’s do a calculation about the effectiveness (Figure 1). The result is striking and the emission brought an enormous impact on environment such as acid rain, global warming and so on.

Figure 1: if the temperature of fermentation raise 1℃，all fermentation industry in China can save nearly 1 billion yuan per year. According to the electric price of industry，1 billion equal to 173310225(kW•h) power. In China, 78% electric energy was produced with coal in 2012. That means we can save 135181975(kW•h) power which made by coal every year. To generate those electricity need consume 50017 tuns of coal! That is a big number! So we can shrift those coal and we can reduce 134776 tuns of carbon dioxide emissions per year.

Figure 2:I’MHeRE include two systems: the customized thermo-tolerance and the intelligent quorum regulating system

Inspired by this, our team managed an intelligent microbial heat regulating engine (I’MHeRE) in E.coli with the methods of synthetic biology. In this engine, two complementary systems (figure 2) were designed. The first one is the customized thermo-tolerance system used to improve E.coli’s heat resistant ability and the other is the intelligent quorum regulating system used for controlling the cell density in an appropriate degree. Moreover, the engine begin to work only when it is properly to do so, ensuring the productive efficiency.

The chassis host with I’MHeRE used in the industry may make the fermentation process less depend on the cooling system and decrease the pollutant emission. On the other hand, cells can live as comfortable as in their optimum temperature, because we extend the because we extend the range of their optimum living temperature and make them live in the optimizing density. Owing to this, the high activity of the enzymes in cell could be kept in a wide range of temperature and the efficiency of microbial metabolism can be improved.

Different strength promoters control different expression level of different heat shock proteins (HSPs) at different temperature to achieve the goal of Hierarchy Heat-resistant.

Figure 3: Hierarchy Heat-resistant.

Figure 4:Overexpression of HSP will saddle the cell growth

We found that overexpressing a protein may affect the growth of the host to a certain degree. With the help of RNA thermometers, they can not only be expressed in a hierarchy way, but decrease the burden of overexpressing HSPs at low temperature. That is the answer why we use RNA thermometers.

Bacteria use complex strategies to control gene expression in response to ambient temperature changing. Many genes encoding heat shock proteins and virulence factors are regulated by temperature sensing RNA sequences, known as RNA thermometers (RNATs), which are located in the 5’untranslated region of the mRNA folding into secondary structure that shed RBS to influence translational efficiency in low temperature. But when temperature is increased to a certain temperature, the RNATS release RBS and the blocked genes express.

Most of gene expression responding to temperature shock measure not the temperature itself but the consequences of temperature-induced damage. Those reaction are effected by temperature indirectly. However, RNA thermometers response to temperature immediately, precisely, and controllably. So using RNA thermometers to control our heat shock proteins expression is a good choice. The RNA thermometers can be available included two types, the natural RNATS and the synthetic RNATS.

Natural RNATS

Figure 6: ROSE RNATS

ROSE family

ROSE RNATS were found in numerous alphaproteobacteria and gammaproteobacteria. All known ROSE elements control the expression of small heat shock genes. ROSE-type structures are composed of two, three or four individual hairpins. The 5ʹ-most hairpins remain stable under heat shock conditions, but the 3ʹ-most hairpin that pairs with the SD sequence is only stable at low temperatures. Temperature-induced local melting in this hairpin exposes the SD sequence, facilitating ribosome binding. The SD region is occluded at 30 °C, partial melting occurs at 37 °C, whereas an increase to 42 °C facilitates the mRNA–ribosome interaction owing to full liberation of the SD and AUG start codon. This type feeds our needs. It may not initiate translation at a certain temperature completely, but the level of expression increase alone with the temperature increasing.

Four U family

Figure 7:the structre of Four U RNATS

Another family of RNATs is a stretch of four uridines that pairs with AGGA in the SD sequence. FourU elements exist in the 5ʹ UTRs of several heat shock and virulence genes. It has two distinct hairpins. The first hairpin is heat stable, however the second hairpin is temperature sensitive, which hide the SD region through the fourU. The formation of the ternary translation initiation complex occurs at high (45 °C) but not at low (30 °C) temperatures.

Synthetic RNA thermometers

Obvious, RNA thermometers provide us superb genetic tools to induce or repress gene expression. However, most natural RNA thermometers are relatively large. They fold into rather complex secondary structures and have been suggested to undergo gradual conformational changes in response to changes in temperature. Therefore, we want to construct smaller and more convenient synthetic RNA thermometers to achieve the goal of hierarchy Heat-resistant regulation. Under the following principle we construct a series of synthetic RNA thermometers.

Stem stability can be influenced by (i) changes in the size and/or GC content of a perfectly matched stem and (ii) introduction of mismatches at different positions.

Figure 8: The structures of Synthetic RNA thermometers simulated using mFold

[1] Nocker A, Hausherr T, Balsiger S, et al. A mRNA-based thermosensor controls expression of rhizobial heat shock genes[J]. Nucleic acids research, 2001, 29(23): 4800-4807.

[2] Neupert J, Karcher D, Bock R. Design of simple synthetic RNA thermometers for temperature-controlled gene expression in Escherichia coli[J]. Nucleic acids research, 2008, 36(19): e124-e124.

[3] Neupert J, Bock R. Designing and using synthetic RNA thermometers for temperature-controlled gene expression in bacteria[J]. Nature protocols, 2009, 4(9): 1262-1273.

[4] Kortmann J, Narberhaus F. Bacterial RNA thermometers: molecular zippers and switches[J]. Nature Reviews Microbiology, 2012, 10(4): 255-265.

[5] Narberhaus F. Control of Bacterial Heat Shock and Virulence Genes by RNA Thermometers[M]//Regulatory RNAs in Prokaryotes. Springer Vienna, 2012: 183-193.

[6] Rinnenthal J, Klinkert B, Narberhaus F, et al. Modulation of the stability of the Salmonella fourU-type RNA thermometer[J]. Nucleic acids research, 2011, 39(18): 8258-8270.

[7] Danino T, Mondragón-Palomino O, Tsimring L, et al. A synchronized quorum of genetic clocks[J]. Nature, 2010, 463(7279): 326-330.

[8] Elowitz M B, Leibler S. A synthetic oscillatory network of transcriptional regulators[J]. Nature, 2000, 403(6767): 335-338.

[9] You L, Cox R S, Weiss R, et al. Programmed population control by cell–cell communication and regulated killing[J]. Nature, 2004, 428(6985): 868-871.

[10] Lila M. Gierasch ,FoldEco: a model for proteostasis in E. coli.Cell Reports, 265–276, March 29, 2012

[11] FU Hartl Molecular, chaperones in protein folding and proteostasis,NATURE, VOL 475, 21 JULY ,2011

[12] Hanna Engelberg-Kulka,Bacterial Programmed Cell Death and Multicellular Behavior in Bacteria,PLoS Genetics,October 2006 ,Volume 2 ,Issue 10

[13] BLOWER T R，SALMOND G P C，LUISI B F． Balancing at survival’sedge: the structure and adaptive benefits of prokaryotic toxin – antitoxin partners［J］． Curr Opin Struct Biol，2011，21( 1) : 109-118．

To verify our work, we find Tianjin team, which have cooperation relationship with us. We verified a part of their work in return. The initial device they designed is BBa_K1020004. During the cooperation,We noticed that the bacteria with this device had a slow growth rate. We thought this was because the leakage of this promoter (J23100) was too strong, for alkR was constitutively expressed and RFP had a high expression level even in the absence of alkanes, the inducible compounds. We assumed that the over-expression of alkR caused negative influence on the growth of cells. To reduce the expression level of the constitutively expressed alkR, eliminating the influence, we designed a modified device BBa_K1020005, and replace J23100 with J23103. It turned out that RFP showed almost no expression without alkanes. If we add C8 as inducible compounds, the red color can be observed and the rate of cell growth is largely increased.

The initial device they designed is BBa_K1020004. During the cooperation, We noticed that the bacteria with this device had a slow growth rate. We think this is because the leakage of this promoter (J23100) was quite strong, for alkR was constitutively expressed and RFP had a high expression level even in the absence of alkanes as inducible compounds. We assumed that over-expression of alkR had a negative influence on the growth of cells. To eliminate the influence, we designed a modified device BBa_K1020005, which replaced J23100 with J23103, in order to reduce the expression level of the constitutively expressed alkR. It turned out that RFP showed almost no expression without alkanes. If we add C8 as inducible compounds, the red colour of the cells can be observed and the growth rate of cells is largely increased.

Figure 1: without alkanes as inducible compounds (BBa_K1020004& BBa_K1020005)

Figure 2: modified BBa_K1020005（with and without alkane as inducible compounds）

https://2013.igem.org/Team:Tianjin/Project/Experiment