Part I - in silico homework

You will design a useful synthetic minimal cell.

1. Pick a function.
2. Design all components that would need to be part of your synthetic cell.
3. Experimental details

An example solution given below, based on: Lentini, R. et al., 2014. Nat comm, 5, p.4012.

1. Pick a function.

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

3. Experimental details

Figure 1 - Artificial cells translate chemical signals for E. coli.

(a) In the absence of artificial cells (circles), E. coli (oblong) cannot sense theophylline.

(b) Artificial cells can be engineered to detect theophylline and in response release IPTG, a chemical signal that induces a response in E. coli.

Part II - Experimental homework

For this week’s experimental homework, you get to test different hypotheses about transcription and translation reactions.

Cell-free transcription

You will design a cell-free transcription reaction where the transcription mix produces fluorescent RNA aptamer called Broccoli, see this work by Filonov et al. The RNA is an aptamer that enhances the fluorescence of a small molecule ligand. Fluorescent RNA aptamers. In the transcription reaction, the Broccoli RNA should fold into a conformation which binds a ligand. The ligand--DFHBI--will fluoresce upon binding to the folded Broccoli. Erkin will set-up your designed experiments for you, following the basic recipe below. He will then measure the aptamer fluorescence increase over time using a 96 well plate to check if the mRNA is successfully expressed in the cell-free system. He will share the data with you and you will interpret the results. The basic recipe of a cell free-transcription reaction is:

Reagents	Intermediate stock concentration	Working stock concentration
Nuclease free water
Transcription buffer	10x	1x
NTP mix	20mM	4mM
Template	50uM	0.14uM
Pyrrophosphatase	10x	1x
Homemade T7	125x	1x
RNAse inhibitor	50	1
DFHBI	100mM	0.1mM

This is a good place for you to generate a hypothesis and for Erkin to test your hypothesis. You can try up to 7 condition (perhaps more if the reagents allow it) plus a positive control. You are encouraged to sample different conditions involving the following antibiotics:

-Kanamycin
-Ampicillin
-Rifamycin
-Puromycin
-Vancomycin
- Dactinomycin
-Chloramphenicol

For example, you might want to sample different antibiotics or different concentrations of select few (0.1X, 1X, 10X, etc)

In addition, you might want to play around with the working stock concentrations of the basic kit. For example, transcription reaction includes Magnesium and Potassium salts. Would too little (or too much) of those hurt your reaction?  You can also test the effects of different pH (by adding a base or an acids into the reaction) or effects of high energy light exposure (i.e, UV light). You might even ask Erkin to spit into a well. Finally, you can also test temperature effects. The key enzyme in this transcription reaction come from a bacteriophage virus which preys on bacteria that like to reside in our bodies. What would the optimal temperature for this reaction be? How would you test that?

Explain in your webpage the importance of each of the reagents you used to transcribe mRNA using the cell-free system. Think about why including controls in your experiments are important and what your positive control in your experimental design would be. Read about antibiotics in general. What makes a molecule an antibiotic? What are the different modes of action of antibiotics? Interpret the results of your experimental results and compare to your initial hypothesis. What would do differently the next time?

Cell-free transcription and translation

First, Erkin will set up a cell-free protein expression experiment using three different cell-free transcription translation systems as shown below. The template (plasmid) is GFP and its spectrum. Once the reaction is setup, Erkin will incubate the reaction at 30C.

1- PURExpress® In Vitro Protein Synthesis Kit from NEB that is prepared roughly according to the protocol based in this seminal Shimizu et al. paper.

2- BioBits®: Central Dogma Kit that is prepared roughly according to the protocol based in this paper by Huang et al. and stored in a -20^oC freezer as a lyophilate for 4 weeks.

3- Homemade cell extract that was stored in a -80^oC freezer for less than a week. This system also requires the addition of energy and amino acid mixtures (that are already present in the other two systems above). These mixtures were prepared roughly according to the protocol based in this paper by Sun et al, while the extract was made by sonication according to protocol based on the this paper by Kwan et al. A typical protein translation reaction with this approach looks like the following:

Tx-Tl Master Mix Formulation (Component)	Example TxTl 15ul Reaction (uL)	35 rxn's of MM (uL)
Mg-glu 1 M	0.18	6.30
K-glu 3 M	0.65	22.75
DTT 0.1 M	0.15	5.25
Energy Mix	1.5	52.50
Amino Acids	1.5	52.50
RNAse Inhinitor A (murine) 40k U/mL	0.3	10.50
T7 homemade (1.5 uM Final) e.g. @ 35 uM Stock	0.643	22.50
Water	3.58	125.30
Cell Extract	5.00	175.00
MM/reaction	13.50	N/A
Template	1.50	N/A

Think and read about the relationship of transcription to translation. Why are these reactions essential to all life called what they are called? Remind yourself the Central Dogma. Explain in your webpage the importance of each of these reagents used to transcribe mRNA and translate proteins using the cell-free system. Come up at least one advantage of cell-free systems over working with live cells. Bet on which of the three systems will work better. Why did you choose one over the other? Think about how ‘working better’ would look like in this experiment. What would be your output from this experiment? How would you quantify it?

Second, you will again have the chance to generate and test different hypotheses here.
For this, we will mainly utilize BioBits®: Central Dogma Kits Try to design your experiments such that you will be testing a hypothesis. You can try up to 7 condition plus a positive control. Again, you are encouraged to sample different conditions. These include the list of antibiotics and conditions mentioned above. If you decide to test the effects of different reaction components refer to the table above which Erkin will set up using the homemade cell extract.

Think about what your positive control in your experimental design would be. Come up with at least one good negative control. Interpret the results of your experimental results and compare to your initial hypothesis. What would do differently the next time?

Please Organize your experiments and send them to Erkin by Friday April 9th following the example format:

	Transcription	Translation
Condition 1	0.1X kanamycin	100X Magnesium
Condition 2	Erkin’s spit	Erkin’s spit
Condition 3	…	No DTT
Condition 4	…	No RNAse inhibitor
Condition 5	..	..
Condition 6	..	…
Condition 7	..	…
Condition 8	Positive control (Which is?)	Positive control (Which is?)

Useful Resources

Databases

• Fluorescent protein database

• Worldwide protein data bank