Overview

title

Traditional fermentation progress uses cooling system to control the temperature, aiming to keep the cells in a good condition. But that result in a great consumption of energy, causing many environmental problems. We set up two artificial systems with the method of synthetic biology to help cells resist heat and control the density of themselves, lightening the burden of cooling system. The devices begin to work only when it is properly to do so, which ensure the productive efficiency. We expect our system to be put into practical fermentation industry in the future.

title
1

How to help the E.Coli resist heat?

There is a protein family called HSP. They can help cells to fix the damage caused by heat by restoring the protein back to normal condition. We implant HSP from hot spring ecosystem into E.coli. As a result, E.coli with our HSP can keep their producing ability, ignoring the hot environment. It can be seen in our customize thermotolerance system.

2

How to control the cell density?

Maybe many people think bacteria are selfish creatures, but that is not totally true. MazEF, is a widely used system among wide bacteria. With this system, many bacteria will sacrifice themselves, and leave nutrition to the left ones when facing threats from dangerous environment, such as heat, lack of food and so on. MazF , which has RNA cleavage activity, is a kind of toxin. MazE, on the other hand, is the antidote of MazF. When MazE combine with MazF, the cell will not be harmed. But once we inhibit the production of MazE, the cell step into programmed cell death(PCD). In our design, we can control the expression of antidote to control the cell density. That is our third device of intelligent quorum regulating system.

3

What means “when it is properly to do so”? And how to fulfill that?

Every new gene is a burden to E.coli. HSPs expressed under a cool condition or extra expressed HSPs are useless, which will disturb normal production progress. We must make control when and how many HSP should be expressed.

We use RNA thermometer as our switch. This kind of RNA has a special stem circle structure, which is opened only when the temperature reach the set status. Then we can make HSP expressed in a hierarchy way.

Similarly, the problem of “when” and “how many” also appears on the control of cell density.

We must ensure that PCD happens when the cell density is high, and the extent must be gentle. Otherwise the cell density might decrease directly and cause negative affect on production. we use quorum sensing device to settle this problem.

Customize thermotolerance system

Bacteria use complex strategies to control gene expression in response to environment 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

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

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.

Based on literature, we know that it’s a practical and effective way to improve E.coli’s heat-resistance by importing exogenous HSPs into it. Heat shock proteins (HSPs) are a group of proteins induced by heat shock, the most prominent members of this group are a class of functionally related proteins named chaperones which are involved in the folding and unfolding of other proteins. The dramatic upregulation of the heat shock proteins is a key part of the heat shock response heat resistance.

div. (From: http://pdslab.biochem.iisc.ernet.in/hspir/chaperone.php)

However, HSPs exist in E.coli can’t improve the heat-resistance much even when being highly expressed. As a result, we paid our attention to other hot-spring bacteria. After several strains were compared, we chose Tengcongensis MB4 which contains abundant kinds of HSPs. Here is a summary:

After further literature, we verified the function of GroEL, GroES, DnaK, DnaJ, TTE, IbpA, FliA, RpoE3 and RpoE7. Here’re the results:

As showed above, GroEL, GroES, DnaK and FliA perform better and we chose these four to construct heat-resistance part.

Quorum-control system

It is obvious that a part of our work aim to control the density. For at the beginning of the fermentation progress the density needs to be improved, it is inappropriate for the density controlling system to run. This density controlling system can be opened only when the cell density is high enough. To settle this problem, we add the quorum sensing device. We use AHL as the symbol of cell density. Cells keep expressing AHL, so that the density of AHL rises with the rise of cell density. There is a kind of protein called LuxR, which can combine with AHL and become a complex. The complex can inhibit downstream gene tetR. The existence of TetR(expressed from tetR) block the oscillation device(wich will be introduced later). As the density of AHL rises, there will be more and more complexes. Then more and more tetR will be inhibited. The oscillation device is activated step by step. The function of this device is to ensure the oscillation device is opened only when the cell density is high enough, so that the oscillation will not block the normal proliferation of E.coli.


Oscillation in different cells is different.In the single cell, the ideal anti-toxin inhibition in PCD device will be like a wave.So the health of individual cell with oscillation device is not effected before the expression of CI, not like constitutive promoter. As a result, some of cell will go apoptosis, and others keep working.

Future plan：
As shown in the title, we are updating the former oscillator into the intelligent variable frequency oscillator.

The C I expressed in oscillation device will activate PCD device. The main part of this device is MazEF system. MazEF system is a toxin-antitoxin system .MazF , which has RNA cleavage activity, is a kind of toxin. MazE, on the other hand, is the antidote of MazF. In nature, many bacteria will sacrifice themselves, and leave nutrition to the left ones With this systemwhen facing threats from dangerous environment, such as heat, lack of food and so on. Before the device is activate, both MazE and MazF are expressed. These substance will combine together to form a hexamer, which do no harm to cells. Once CI is expressed, the expression of MazE will be inhibited. No more hexamer will be formed, and MazF will show it’s power. Because of mazF, the density of cell will decrease. When the density is too low that cannot activate quorum sensing system to activate oscillation system, there will be no CI. After that, the MazE will be expressed again, which will save the cells from the hand of MazF.

Parts

1

New Parts

BBa_K1117001
DnaK

DnaK is one of the Hsp70 proteins expressed in prokaryotes. By binding to partially peptide sequences, it can prevent proteins from aggregating and being rendered unfunctional.

http://parts.igem.org/Part:BBa_K1117001

BBa_K1117002
GroEL

GroEL(Hsp60) belongs to the chaperonin family of molecular chaperonescan. It could help proteins to fold properly with the presence of protein complex GroES(Hsp10).

http://parts.igem.org/Part:BBa_K1117002

BBa_K1117003
GroES

GroES(Hsp10) is also known as chaperonin 10 (cpn10) or early-pregnancy factor (EPF). It could help proteins to fold properly with the presence of GroEL(Hsp60).

http://parts.igem.org/Part:BBa_K1117003

BBa_K1117004
ThiF

ThiF acts like an E1 ubiquitin-activating enzyme for ThiS, which is a ubiquitin-like protein in prokaryotes. The process of ubiquitination can target denatured proteins and stimulate their degradation.

http://parts.igem.org/Part:BBa_K1117004

BBa_K1117005
J23119-K115001-DnaK

A strong constitutive promoter J23119 linked to the 42℃ RNA thermometer K115001 and heat shock protein DnaK K1117001.

http://parts.igem.org/Part:BBa_K1117005

BBa_K1117006
J23119-K115001-GroEL

A strong constitutive promoter J23119 linked to the 42℃ RNA thermometer K115001 and heat shock protein GroEL K1117002.

http://parts.igem.org/Part:BBa_K1117006

BBa_K1117007
J23119-K115001-ThiF

A strong constitutive promoter J23119 linked to the 42℃ RNA thermometer K115001 and enzyme ThiF K1117004.

http://parts.igem.org/Part:BBa_K1117007

BBa_K1117008
J23110-K115002-DnaK

A medium strength constitutive promoter J23110 linked to the 37℃ RNA thermometer K115002 and enzyme DnaK K1117001.

http://parts.igem.org/Part:BBa_K1117008

BBa_K1117009
J23119-K115001

A strong constitutive promoter J23119 linked to the 42℃ RNA thermometer K115001.

http://parts.igem.org/Part:BBa_K1117009

BBa_K1117010
J23110-K115002

A medium strength constitutive promoter J23110 combined with 37℃ RNA thermometer K115002.

http://parts.igem.org/Part:BBa_K1117010

BBa_K1117011
J23119-B0034-C0062-B0015

We change the Constitutive Promoter II of BBa_I9020 to make it more efficiency to feed our need.

http://parts.igem.org/Part:BBa_K1117011

BBa_K1117012
I719005-P0451

Inducible promoter T7 controls the expression of CI.

http://parts.igem.org/Part:BBa_K1117012



2

Used Parts

NAMEDESCRIPTION
J23117Promoter
B0034 RBS
C0062luxR repressor/activator
B0010Terminator
B0012Terminator
J23103Promoter
C0061Autoinducer synthetase for AHL
R0063Promoter
C0040tetracycline repressor from transposon Tn10 (+LVA)
C0012lacI repressor from E. coli (+LVA)
C0051cI repressor from E. coli phage lambda (+LVA)
R0051Promoter (lambda cI regulated)

Cooperation

Safety

1

Organism & Experiment safety

The organisms used in our experiment conclude:

  • Escherichia coli BL21(DE3)
  • Escherichia coli K12

Although all of the organisms are in line with biosafety level 1 (BSL), we have treated it as a potential affection source and created following rules to minimize the danger:

  • Wearing appropriate protective equipment like gloves and lab coats.
  • Don’t eat any food or store it in the lab.
  • Avoid wearing shorts or shoes that make your legs or toes exposed.
  • Washing hands before leaving the laboratory.

2

Safety training

There is no Biosafety Committee at our university, only a bio-safety officer. Nevertheless, we have iGEM instructors, Dr. Chun Li and Dr. Shuyuan Guo, who trained us about the project progress regularly and is responsible for the bio-safety of our project. The general rule at our university is that every student has to take part in specific safety training before their first experiment. Further, once a year, every member of the lab is required to take a refreshment course in lab safety, bio safety, fire safety, waste management, handling gas cylinders and working with hazardous materials. All of these treatments ensure our lab safety.

3

Public & Environment safety

The model organisms used in our project are non-virulent strains which are commonly used in microbiology laboratories. The organism has been provided various resistance of antibiotics such as chloramphenicol, ampicillin and kanamycin. Without the circumstance which contained specific antibiotic, our organism has no advantage in the competition with the wild one. Besides, although we have improve its resistance of higher temperature(42~45℃), it can still be denatured via heat. In a word, it can work well in laboratory environment and will be eliminated through competition when exposing in the wild.