Synthetic Minimal Cells

Assignment

Theory Homework : Design an useful synthetic minimal cell.

Design a system that can perform a function (biosensor, biomanufacturing, research - or any other purpose). Design all the steps of building and deploying the system: define function, pick components, describe results. Example solution to the SMC assignment, based on: Lentini, R. et al., 2014. Integrating artificial with natural cells to translate chemical messages that direct E. coli behaviour. Nature communications, 5(May), p.4012.This paper is among the files I put in this google drive folder. The example answers are given in italic. To complete the homework, answer all questions.

1) Pick a function.

1A) What would your synthetic cell do? What is the input and what is the output.
Conceptual Idea

I would like to make fart disassemble or deodorize system by using SMC. For example, when we put fart in aquariusthe water carrier which included E.Coli who has artificial cell with into culture medium, this E.Coli has artificial cell who has bio sensor to make pore after detecting Indole - from "The fart studies" which make stink. And after this detecting, make protein which functions to disassemle or deoforize indole. Then, it removes the stink into this aquariusthe water carrier. Without Indole, Ecoli does not make odor protein.

9% gas of a fart is gas called nitrogen, oxygen, hydrogen, carbon dioxide, and methane
which occupies 99% does not have a smell. The stink is only 1% or less of gas. Ammonia, Hydrogen sulfide, Indole, Skatole.
These gas is produced when the bacteria in the large intestine disassemble protein.
  • Input:Indole
  • Output of the SMC: Some materials function as signal of protein like as isoamyl alcohol which is chemical precursor.
  • Output of the whole system: Odor protein produced in bacteria like as banana smell protein - .
  • Reference : IGEM HP
Memo : Primary points in order to make SMC
  • Induce chemical reaction by using some material riboswitch.
  • This chemical reaction is not detect by Ecoli it self.
  • After detecting chemical material, artificial cell makes reaction in order to get some promoter and induce protein production in E.Coli.
Memo : Why use IPTG in SMC and out input will be GFP in produced bacteria?How theophyline fuctions?
  • The artificial cell is built with a phospholipid vesicle containing isopropyl b-D-1-thiogalactopyranoside (IPTG), DNA, and transcription–translation machinery. The DNA template codes for a previously selected riboswitch that activates translation in response to the presence of theophylline10.
Memo : How can I choose the function like as theophyline? What can I refer riboswithch like as theophyline??
  • Refer to this reading. Theophyline Aptamer reference: Martini, L. & Mansy, S.S., 2011. Cell-like systems with riboswitch controlled gene expression. Chemical Communications, 47(38), p.10734.
  • Important points are expression of protein will be control by riboswitch like as theophylline.
Structure
1B) Could this function be realized by cell free Tx/Tl alone, without encapsulation?
  • No. If the isoamyl alcohol is not encapsulated, synthetic cell actuator would not exist. Therefore Banana oder will not be happened and Indore smell is as it is.
1C) Could this function be realized by genetically modified natural cell?
  • I think..it might be possible if indole aptamer could be incorporated into gene..
1D) Describe the desired outcome of your synthetic cell operation.(Example : In presence of SMC, bacteria sense theophylline.)
  • In presence of synthetic minimal cell, becteria sense Indole.

2) Design all components that would need to be part of your synthetic cell.

2A) What would be the membrane made of? (Example : Phospholipid + cholesterol.)
  • I am not sure what kind of components are appropriate.. But it might be phospholid.
2B) What would you encapsulate inside? Enzymes, small molecules. (Example : Cell free Tx/Tl system, IPTG, gene for membrane transporter under the control of theophylline aptamer)
  • This also not sure.. But I guess gene for memembrane tanspoter under the control of Indole aptamer..
2C) Which organism your tx/tl system will come from? is bacterial OK, or do you need mammalian system for some reason? (hint: for example, if you want to use small molecule modulated promotors, like Tet-ON, you need mammalian system.
  • May be becterial..
2D) How will your synthetic cell communicate with the environment? (hints: are substrates permeable? or do you need to express membrane channel?

The membrane is permeable to the input molecule (Indole), the output is isoamyl alcohol that will cross the membrane via the membrane pore created after Indole sensing.

3) Experimental details

3A) List all lipids and genes (bonus: find the specific genes; for example, instead of just saying “small molecule membrane channel” pick actual gene)

It might be..

  • These parts )
  • Isoamyl alcohol
  • Biological cells: E.coli with transformed Gene
  • Indole aptamer..
3B) How will you measure the function of your system?

One person put fart into aquariusthe water carrier. And another person close the carrier. If this system work properly, odar should be banana. But if not..stink...

Lab homework assignment: Express GFP in cell free system.

The kit

  • enzyme mix at 1.33x
  • plasmid P70-deGFP from Vincent Noireaux, same as used here: http://pubs.acs.org/doi/abs/10.1021/sb200016s OR other GFP expression vector under endogenous E.coli promotor like sigma 70.
  • Please keep the enzyme mix frozen at all times - in -80 freezer or supplementing the dry ice until you're ready for experiment.
Memo : Vocabulary
  • carboxy terminus : C terminus. Final destination of protein coding frame..
  • Riboswitch : It is one part of mRNA. This pars are related to expressing protein like as switch on and off. For example, this mRNA detects volume of vitamine in molecures and adjust expressing of protein.
  • αHL: α-hemolysin, a kind of protein.
  • phospholipid :リン脂質
  • Pore : 孔 - go throgh menbraine and penetrates materials
  • Aptamer : Oligonucleotide or peptide molecules that bind to a specific target molecule.

Weekly Reflection:

Do your activities this week raise new ethics and/or safety considerations you had not considered in week 1? Describe what activities have raised these considerations and any changes you have implemented in response.

  • Bio sensor seems conditional branch system organized by organism although it is not related to safety ethic..It is very interesting.

Memo : Reading "Integrating artificial with natural cells to translate chemical messages that direct E. coli behaviour" (DOI: 10.1038/ncomms5012 | www.nature.com/naturecommunications)

1. What is this research?
  • Previous efforts to control cellular behaviour have largely relied upon various forms of genetic engineering.
  • However, other methods of cellular control are possible.
  • The artificial cells expand the senses of Escherichia coli by translating a chemical message that E. coli cannot sense on its own to a molecule that activates a natural cellular response.
2. What is the great finding comparing previous research?

-The team instead construct artificial cells that could interact with natural cells in order to evoke a behavioural response. The artificial cells in this system function as chemical translators that sense molecules that E. coli alone cannot sense. -In response, the artificial cells release a molecule that E. coli can naturally respond to, thereby translating an unrecognized chemical message into a recognized chemical message.

3. What is the most important factors of this technology(method)?

-The artificial cell is built with a phospholipid vesicle containing IPTG, DNA, and transcription–translation machinery . The DNA template codes for a previously selected riboswitch that activates translation in response to the presence of theophylline10. The theophylline riboswitch controls the synthesis of the pore forming protein a-hemolysin (aHL). Therefore, in the presence, but not the absence, of theophylline a pore forms that releases entrapped IPTG.E. coli alone does not respond to theophylline,and IPTG does not cross the vesicle membrane of the artificial cell in the absence of the pore. -The ability of E. coli to receive the chemical message sent by the artificial cells is assessed in two ways. First, the fluorescence of E. coli carrying a plasmid encoding a fluorescent protein behind an IPTG-responsive, lac operator sequence is evaluated. Second, the gene expression of untransformed E. coli is monitored by reverse transcription quantitative PCR (RT–qPCR).

4. How this research verify the result?

-The theophylline-sensing device is functional in vitro. To build artificial cells that sense theophylline and in response release IPTG. -a theophylline-sensing genetic device was built with a T7 transcriptional promoter, a theophylline riboswitch and a gene encoding a fusion between aHL and super folder GFP at the carboxy terminus. -Finally, K30S E31T aHL- GFP was placed behind the theophylline riboswitch to test the activity of the cell-free sensing device. A clear difference was observed between protein expression in the presence and absence of theophylline

-Direct genetic engineering of living cells is not needed to control cellular behaviour. It is possible, instead, to coerce desired activity through communication with artificial cells.

Memo Reading : "Cell-like systems with riboswitch controlled gene expression" (DOI: 10.1039/c1cc13930d)

1. What is this research?
  • Synthetic riboswitches can be used to control protein expression under fully defined conditions in vitro
2. What is the great finding comparing previous research?
  • One class of regulatory RNAs, known as riboswitches, is located within the untranslated regions of mRNA. In bacteria riboswitches can up or down regulate gene expression in response to ligand binding directly to the mRNA.
  • this assay show that a synthetic theophylline riboswitch requires no additional components for activity beyond a T7 RNA polymerase and the minimal E. coli translation machinery.
3. What is the most important factors of this technology(method)?

-The presence of an extravesicular ligand converts the cell-like system from the OFF-state to the ON-state. -RNA is transcribed from DNA . RNA is only translated into protein in the presence of the activator ligand, which in this case is theophylline.

4. How this research verify the result?

-Both riboswitches were inserted into the 50-untranslated region of sequences encoding the yellow fluorescent protein YPet.11 The genetic constructs also included a T7 transcriptional promoter and an E. coli ribosome binding site. -Riboswitch activity with minimal transcription and translation components was monitored in real time by fluorescence spectroscopy. Little protein expression occurred in the absence of theophylline for the synthetic riboswitch system, whereas protein levels were 6-fold higher after 6 h in the presence of theophylline.

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