Bio 123, UZH

• The synthetic network (repressilator) works as oscillator
• However, the oscillations are not regular
• In contrast, natural oscillators are very precise
• Nature is using different network designs

The toggle switch was built with 2 repressors and can be switched between 2 states

The repressilator consists of 3 repressors and oscilates

Its the elementary building block of a digital circuit.

Each gate has a separate symbol and a name, f.e. "A or B", "not A", etc.

The relationship between the in- and outputs is given in the truthtable. Its always either 1 or 0. (zero means for example that we have no input of A)

Here you will need both together (A and B) to have an output. One alone is not enough. 

There are also layered AND gates, when you combine different gates together.

An example of an AND gate is the treatment of Psoriasis. Its a chronic inflammatory skin disease.

Gates can also be built by using integrases.

With our input we control these integrases. They can for example inverte the terminator, or even cut the whole thing out!

This one works when either A or B are present

see picture

In the classic cloning, you have a cDNA library, which you amplify by PCR, etc.

But nowadays, you just go online and order your genes of interest. And they synthesize the DNA for you. There are many advantages (you can delete restriction sites etc.) and its not even that expensive.

But how do they synthesize these genes for you?

Take the DNA, divide it into parts and synthesize Oligonucleotides which have overlappings to each other. Then insert primers with uniques sites.

1.       Many parts are undefined

2.       The circuits are unpredictable

3.       Many parts are incompatible

4.       Variability crashes the system

5.       Safety and ethical challenges

Genetically identical cells which are grown under same circumstances and still show differences

Its logical that individual cells from an organism are different (f.e. From the liver, heart, etc.).

But how about the variability between cells from the same type, that are siblings from the same parent cell and are grown under identical conditions?

And where lies the boundary between differentiation and non-genetic cell-to-cell variability?

(Its to mention that to notice this is only possible with the newest technologies, where we can quantify the cells)

Such variability cannot described with qualitative terms, so we need quantification.

• Fluorescence activated cell sorter/analyzer (flow cytometry)
• Single-cell micro-dissection for further analysis

• Ultrasensitivity
• positive feedback

Intrinsic noise is when you have two gens which are regulated identically, but you get different outputs (even under identical conditions!).

Otherwise, when you get the same output (and maybe sometimes a little darker yellow or lighter, etc.) you only would have extrinsic noise.

Intrinsic noise is when you have two gens which are regulated identically, but you get different outputs (even under identical conditions!).

Otherwise, when you get the same output (and maybe sometimes a little darker yellow or lighter, etc.) you only would have extrinsic noise.

when you cannot explain where it cames from, its stochastic, not regulated or controlled

Active: can measure noise and somehow create an anti-noise signal which cancel the noise out

Passive: prevents noise from entering 

Active: can measure noise and somehow create an anti-noise signal which cancel the noise out

Passive: prevents noise from entering 

Certain controllers, measure something, have an output,

Feed-back/forward motifs

But the problem with this one is that it cost energy!

Builiding a compartiment which shields it off from other compartments(compartimentize the reaction, doesnt cost much energy)

• Highthroughput-sequencing technologies parallelize the sequencing process producing  thousands or millions of sequences at once

• Low cost production of large volumes of sequence data

A collection of DNA fragments with adapters ligated to each end

The stretches of DNA read by the sequencer (the library consists in the end of reads)

Identifier sequence that allows pooling several samples without physical separation

(Barcoding: DNA-Barcoding (englisch DNA barcoding) ist eine taxonomische Methode zur Artenbestimmung anhand der DNA-Sequenz eines Markergens.)

Deutsch: Angleichung/Anpassung

The aligning and merging of reads in order to restruct the original sequence

You can distinguish two types:

1. Mapping= assembling reads against an existing backbone sequence
2. De novo=  assembling short reads to full length sequences

E: Sequenced reads that overlap are reassembled back into longer sequences.

D: Ein Contig ist ein Satz überlappenderDNA- oder Protein-Stücke (reads), die von derselben genetischen Quelle stammen. Ein solches Contig kann dazu genutzt werden, die Original-DNA-Sequenz dieser genetischen Quelle abzuleiten.

A series of linked contig which are in the right order but not necessary connected in one continuous stretch of sequence

Either how many or how long reads we create

The average number of reads representing a given nucleotide in the reconstructed sequence is called depth of coverage

See picture

- High number of reads and low input

- Short read but very high throughput

- clonal amplification

- State-of-the-arts optics used for detection

- At the end of the bridge amplification, you get a cluster. This is what you actually going to sequence!

- SBS (sequencing by synthesis)

- Mid-to-High number of reads and low input

- Short reads and low throughput

- but very quick (usefull during a surgery when quick results are needed)

- beads (Kugel) are used for monoclonal amplifications

- ph-meter is used

- no modified nucleotides

- Low number of reads and high input

- SMRT (single molecule real time sequencing)

- Eavesdropping (lauschen) on the DNA polymerase as it replicates DNA 

- Only to the last phosphore ist fluorescent dye attache, the strand is natural

-> https://www.youtube.com/watch?v=v8p4ph2MAvI

- Low numper of reads and high input

- sequencing of long fragments

- flow of ions that are transported through perforated membranes

- https://www.youtube.com/watch?v=3UHw22hBpAk

1.       Forward Genetics

This is the more classical approach. Here you do random mutagenesis and then screen for cells with a change in the phenotype. So you select for a phenotype, and then identify the mutated gene.

2.       Reverse Genetics

This is really the opposite, I already know which gene I want to test, and what mutant, but in the past we couldn’t really do that because we didn’t know all genes.

So here you start with a gene of unknown function (but you know exactly which gene it is), then you do (only with this gene!) an overexpression or another sort of mutation. Then you examine the resulting change in the phenotype to infer the function.

-> "targeted" mutagenesis

- Grow a very large number of cells in a dish,

- Then you throw in an agent who randomly mutate some cells, but limited so you get one or even less mutation per cell! (=limited random mutagenesis)

- Next step is to select the cells with the desired phenotyp. There are two ways you can do that:

Either by testing the resistance (throw something on the cell that kill all the cells BUT the one with mutations, for example toxic compound or a virus.) or second possibility by a fluorescent reportert (you can mark either type, the normal ones or the ones with a mutation)

The last step is to identify the mutation by sequencing.

-First generate a large number of reagents each perturbing a specific gene. (3 important methods: cDNA, RNAi, CRISPR-CAS9)

- Then make an arrayed (geordnet) library where each reagent has a pre-defined position (which is called a "well"). Third step is to add cells to each well and systematically score for phenotypes. Last, you assign the function to each gene.

cDNA = copy DNA from mRNA

You use reverse transcriptase to get a DNA from a mRNA. Because the mRNA is already processed (e.g. Spliced) there are also no introns etc.