Team:NCTU Formosa/project

Temperature-regulated system

Introduction

It is not a dream anymore that "making the impossible possible" with the increase of modern technology. It is also a serious problem that what human has brought to Earth along with our civilization. Drastic environmental changes are inevitable due to the fact of massive developing industries. Temperature changing are one of the problems we need to confront.

Human cells are constantly subjected to changing environmental conditions. Comparing with chemical substances, temperature is more accessible, convenient and lower costing. Moreover, temperature is also less burden to the organisms. Therefore, few organisms use temperature as a regulating factor. A mechanism found in different organisms that makes the cell respond to thermal changes, for example, is the RNA thermometer.

An RNA thermometer is a temperature-sensitive non-coding RNA molecule which regulates gene expression. The expression of heat-shock, cold-shock and some virulence genes are coordinated in response to temperature changes.There are several systems suggested in references that are based on RNA secondary structure. This structural transition can then expose or occlude important regions of RNA such as a ribosome binding site, which then affects the translation rate of a nearby protein-coding gene, so we can say that the RNA thermometers are just like riboswitches.

Apart from protein-mediated transcriptional control mechanisms, translational control by RNA thermometers is also a widely used regulatory strategy.

Mechanism

RNA thermometers are identified in the 5' UTR of messenger RNA, upstream of a protein-coding gene. They are able to occlude the ribosome binding site (RBS) and prevent translation of mRNA into protein.

Unlike normal RBS, the 37°C RBS has a unique or rather stable hairpin structure which shaped like a lock. If the temperature drops below 37°C, the base-pairing neucleotides on the RNA will form a stable secondary hairpin structure, occluding the Shine-Dalgarno sequence thus disabling the ribosome to bind. The base-pairing of this RNA region will block the expression of the protein encoded within. Since the structure is sustained by base pairing, heat can be employed to break the hydrogen bonds. So when it reaches 37°C , the bonds will be broken and the hairpin would be unfolded, causing the Shine-Dalgarno sequence to be exposed and permitting the binding of the ribosome, which then starts the translation. This means that by raising temperature to a certain threshold, the 37°C RBS can function as a normal RBS. In this way, gene expression can be regulated on the RNA level by temperature.

Design

An RNA thermometer in the 5'-UTR of the Salmonella agsA gene, which codes for a small heat shock protein that can be used for temperature sensitive post-transcriptional regulation, initiating translation at 37°C. Transcription of the agsA gene is controlled by the alternative sigma factor σ32. Additional translational control depends on a stretch of four uridines that pair with the SD sequence, so they are named "FourU". They repress translation of this protein by base-pairing with the Shine-Dalgardo sequence of the mRNA. This prevents ribosomes from binding the start codon.

We used the RNA thermometer to build up a low-temperature released system to control the expression of targeted protein. A specific ribosome binding site (RBS) BBa_ K115002 with high translation activity at high temperature(> 37°C) and low translation activity at room temperature was used to design the temperature-dependent genetic circuit in E. coli, with a green fluorescent protein (GFP) used as the reporter protein.

In our project, we use this 37°C RBS in both red light induced circuit and dark response circuit and it is related to the production of GFP and BFP. We use this 37°C RBS to design our project because we want to use different conditions to produce different substances. Therefore, the characteristic and sensitivity of 37°C RBS is just fit in our purpose which we want to multiple regulate our system through temperature.

Reference

Small RNA-regulated system

Introduction

Base pairing offers a powerful way for one RNA to control the activity of another. Both prokaryotes and eukaryotes have many cases which a single-stranded RNA base pairs with a complementary region of an mRNA. As a result, it prevents expression of the mRNA.

RNA interference(RNAi), also called post transcriptional gene silencing, is a process in which RNA molecules inhibit gene expression by destroying specific mRNA molecules. In 2006, Andrew Fire and Craig C.Mello shared the Nobel Prize in Physiology or Medicine because of their study on RNA interference¹¹. This powerful gene silencing tool in eukaryotes has been used in many research. As their counterparts in bacteria, small non-coding RNAs(sRNAs) are important regulatory roles.

Small RNAs (sRNAs) have become increasingly significant in playing the role of bacterial gene regulation. Most sRNAs interact with the targeted mRNAs by imperfect base pairing, reducing the translation efficiency and recruiting chaperones such as Hfq for translation termination. The sRNAs would bind on the target with some of it's own sequences wind to create hairpin-like structure, and the chaperons would stuck between the hairpins in order to protect the sRNA-mRNA combination from degrading. In the end, sRNAs regulate gene expression by forestalling translation. It is an effective and specific regulated-system that functions under RNA level. Since this regulated-system has already been employed in vivo, it is necessary to design artificial sRNA that targets specifically to desired genes. This way, we can prevent the sRNA from affecting undesired genes.

Unlike the regulated-system popular used in iGEM projects(e.g., P_tet & tetR, P_lux & LuxR, etc.), sRNA regulated-system seems more efficient. Because the inhibition works under RNA level, which means the energy wasted in E. coli is less than other system(producing proteins to regulate).

Figure 8 depicts how the small RNA chaperone, Hfq, associates with the small RNA and represses a target mRNA.¹²

Mechanism

How the small RNA regulate gene expression? Figure 9 shows the mechanism of repression of one target mRNA. The small RNA has three stem-loop double stranded RNA structures, and the loop closest to the 3’ terminus is complementary to a sequence preceding the initiation codon of target mRNA. Base pairing between small RNA and target mRNA prevents the ribosome from binding to the initiation condon, so the translation would be repressed.^13,14

Design

We have designed two artificial sRNA each modify from different paper.The sRNA-1 is based on afsRNA ARlacZ1,which is created by Hongmarn Park and his colleagues.¹⁵ SibC is a small RNA sequence which is already found in E.coli. Hongmarn Park and his colleagues change few A-U base pairs of SibC into G-C base pairs to make ARlacZ1,in the purpose to let the combination of the artificial RNA and its target more tight. After then, they try to change to 9 different location of target-recognition sequence to test the effect on gene silencing.Our design is modify from the RNA sequence which has the best effect: ARlacZ7. In accordance with afsRNA ARlacZ7, we change the target-recognition sequence to finally complete Our sRNA-1. We also use another

記得下面補兩個sRNA的sequence 和結構圖哦

The sRNA employed in this project was picked from a library of artificial sRNA that was constructed by fusing a randomized antisense domain of Spot42, the scaffold that is known to recruit the RNA chaperons. The sRNA we picked contains a consensus sequence, 5’-CCCUC-3’, that can base pair with the SD sequence due to complementarity. This sRNA, as expected, effectively regulates gene expression by reducing translation efficiency and recruiting Hfq. In addition, this sRNA shows high specificity against its targeted gene, ompF, as it doesn’t hold significant activity against other genes from E. coli genome (Sharma and others, 2011).

The sRNA picked is competent, but we hoped that it can target any desired gene. With that said, we designed a RBS by employing the sRNA targeting region from ompF and making the AUG codon sufficiently apart from the SD sequence for ribosome binding. By adding this RBS to the upstream of any desired gene, the gene can be regulated by sRNA.

Reference

Mechanism of the E.colightuner

Introduction

This is the pathway we designed by using the three different regulating factors. We tend to set up four conditions for the E.coli to work by paring the light and the temperature controls. Distinguished by whether red light exists or not, the pathway is divided into two parts, the Red Light Induced Circuit and the Dark Response Circuit. We use four different fluorescent proteins as reporter genes to test that whether the switch of our pathway is successful.

Red Light Induced Circuit

At 30°C and under red light, Pred is activated for translation proceeding. However, ribosomes can only bind to the normal RBS, as 37°C RBS (tRBS) forms a hairpin structure, forestalling ribosomes from binding. The only gene downstream of the normal RBS is RFP, so only RFP would be expressed.

On the other hand, with red light, 37°C RBS unfolds at 37°C , resulting in the expression of both LuxR and GFP. LuxR would bind with AHL(converted by LuxI) to form a complex that activates Plux. The activation of Plux produces sRNA that binds to the normal RBS on mRNA level, blocking it from ribosomes. As a result, RFP would not be expressed and only GFP remains at 37°C.

Without red light, Pred would not be activated, and therefore, the red light induced circuit is completely shut down in the dark.

Dark Response Circuit

Pred activates in the presence of red light, producing tetR that represses Ptet. In other words, the dark induced part is inactive in the exposure to red light and active without red light. At 30°C in the dark, only normal RBS functions to express YFP while 37°C RBS remains as a hairpin structure.

At 37°C, however, 37°C RBS unfolds to express LuxR and BFP. Just like the mechanism employed in the light induced part, LuxR forms a complex with AHL to activate Plux that produces the sRNA to block normal RBS. This way, the YFP downstream of YFP cannot be expressed and BFP is expressed at 37°C.

Summary

This is the pathway summary of the E.colightuner. There is one kind of fluorescent protein in each condition we set. It means that we can control four gene expressions by using the E.colightuner. The only thing we have to do is changing the different environmental conditions for specifically expression.

Future Work

The multiple regulated-system we have created in this project consists of two different parts: light induced part and dark induced part. Each part regulates two different genes, resulting in a total of four genes that can be regulated. By adding new parts to the system, it is plausible that the system can regulate more genes. We tend to do that by employing more light sensing promoters. By adding each light sensor, we would be able to regulate two more different genes by employing regulation mechanism of 37°C RBS and sRNA.

Application

Taiwan is renowned for Phalaenopsis, producing more than 35 million Phalaenopsis each year. There is no doubt that Taiwan has become "The Kingdom of Phalaenopsis". The technique we usually use to grow Phalaenopsis is plant tissue culture, thus, we intend to apply our system to plant tissue culture as well.

Tissue culture is the growth of tissues or cells separated from the organism. This is typically facilitated via use of a liquid, semi-solid or solid growth medium, such as broth or agar. Moreover, hormones are added to the growth medium with a view to meet what plants need during growth period. However, the hormones needed during each growing state are not the same. So, we need to figure out a device which can generate multiple hormones. By adding our engineered E.coli to the growth medium, the plants can gain multiple hormones secreted by E. coli under different circumstances. We chose four common hormones as our genes, Auxin, Cytokinin, Gibberellin, and Florigen.

Since Florigen(FT protein) is the protein which isn't produced by E.coli naturally, there isn't any secretion system in wild type E. coli to transport Florigen outside of the cells. Though secretion by cell lysis is a possible method to solve this problem, we still seek for other ways in order not to let our E. coli die during the secretion process. Thus, we ought to create a secretion system which can help Florigen pass through the inner and outer membrane of E. coli. We have founded a system from 2012 Kyoto iGEM team called Twin Arginate Translocation (Tat) pathway, which can help sending Florigen through the inner membrane to periplasm. In Tat pathway, a Tat transporter recognizes TorA signal, and proteins having a TorA signal at their N terminals can get into the periplasm. Moreover, 2012 Kyoto iGEM team also found out that kil protein from λ phage can make holes on outer membrane, which then transporting Florigen outside of cells. In a word, with kil protein gene and the fusion of torA and FT protein gene in our biobrick, we can successfully transport Florigen out of the E. coli.

The use of Auxin is to induce the growth of shoot, but the excess amount will kill the plant. We want to reach the great amount which will promote the plant’s growth but not to kill the plant. After researching, the Imperial College London 2011 iGEM team also has the same problem. The following is their solution. They chose the native Auxin indole-3-acetic acid(IAA) to use, since the synthetic Auxin is more stable that it can live for weeks. IAA also exist the risk of toxic amount. To ensure the sufficient but not excess amount to promote the growth of shoot, they give a model to reach the greatest amount.

We intend to carry out our application with the device beneath. Cultured in the growth medium circulation, our engineered E. coli can absorb enough nutrition to express the desired genes properly. We also use a semi-permeable membrane, which allows hormones but not bacteria themselves to pass through, separating the bacteria from plant tissues. Thus, we keep the tissue culture medium sterilized.

Under red light and 37°C, Cytokinin will be produced. Cytokinin is a plant growth substance which can primarily induce cell growth and differentiation in plant roots and shoots. It can decrease apical dominance which will make the main stem grow better compared to other stems. In this way, all the stems are able to become stronger. Cytokinin can also signal lateral bud growth, which will make the plant bushier.

Under red light and 30°C, Auxin will be produced. Auxin can coordinate development from cellular, organs to the whole plant. Auxin molecules in cells may cause direct responses by stimulation and inhibitions of certain gene expressions. On the cellular level, Auxin is important for cell growth. It can induce axial elongation in roots and lateral expansion which can make the roots swell. For the whole plant level, it affects the shape of the plants. Because of the different functions, each organ has uneven Auxin concentration and causes specific shapes.

Under dark and 30°C, Gibberellin will be produced. Gibberellin is important in the elongation of cells. It causes the extension of stems by inducing cell divisions and elongations. A special characteristic of it is that it can produce more mass when the plant is exposed to cold temperature.

Under dark and 37°C, Florigen will be produced. Florigen is the key hormone to trigger plant blossoming. We use a promoter that can promote transcription of the FT gene, which then translate into FT proteins. FT proteins will then be transported via the phloem to the shoot apical meristem, where it will interact with the FD protein(transcription factor) to activate floral identity, thus inducing flowering.

The next big thing

On the other hand, we tend to optimize our system to achieve subtle control of the four gene expressions. We might be able to do this by taking the values between the limits we have set. Instead of 30°C and 37°C , we tend to take the values between, so we can create more conditions under which different level of expressions can be achieved. We can also vary the intensity of red light to control the level of expression. For instance, we can get different pigment outputs by tuning the temperature and varying the intensity of red light. The ultimate goal is to precisely control the level of expression of each gene.

因為Pred的直接性，在未來可以拿來測試被紅光、藍光的影響度?

Reference