Other important stuff

Question 1

How do we grow and propagate Thalassiosira cells?

Answer 1

Thalassiosira cells that were growing exponentially were harvested by centrifugation. Then the supernatant were moved, and the cells were moved into another tube and kept on ice for the further work. The cells were spun down again, and the supernatant were removed. The cells were resuspended in sorbitol, and then washed in sorbitol twice. Plasmid DNA and ssDNA were then added to the cells. The mixture was incubated on ice, and then electroporation were performed.
The cells were subsequently placed in L1 medium and incubated for 24 hours at constant illumination at 18-22*C for recovery. The culture was then centrifuged and split into two. One half were plated onto 5 plates, while the rest was kept in L1 medium. The clones were picked 4-7 days and cultivated in L1 medium. Colonies were counted after 14 days.

Question 2

What are the elements included in the plasmid vector for the transformation, and what are their functions?

Answer 2

(1) Lac promotor: the promotor is a DNA sequence which proteins (RNA polymerase) binds to initiate transcription of a single RNA transcript from the DNA downstream of the promotor.

(2) Lac operator: an operator is a segment of DNA to which a repressor binds. It is located between the promotor and the genes of the operon.

(3) U6 3´UTR: this region is best known for regulating mRNA-based processes, such as mRNA localization, mRNA stability, and translation.

(4) PAM: PAM is also known as “the protospacer adjacent motif” and is a short specific sequence following the target DNA sequence that is essential for cleavage by Cas nuklease. PAM is about 2-6 nucleotides downstream of the DNA sequence targeted by the gRNA, and the Cas cuts 3-4 nucleotides upstream of it.

(5) Cas9: Cas9 - one of the enzymes produced by the CRISPR system - binds to the DNA and cuts it, then shutting the targeted gene off. Modified versions of Cas9 can be used to activate gene expression instead of cutting the DNA.

(6) HIS3 promotor: the promotor is a DNA sequence which proteins (RNA polymerase) binds to initiate transcription of a single RNA transcript from the DNA downstream of the promotor. HIS3 encodes an enzyme involved in histidine biosynthesis.

(7) AmpR promotor: the AmpR promotor is the promotor of ampicillin resistance.

(8) Ori: the origin of replication is the initiation site for plasmid DNA replication recognized by the bacteria.

Question 3

What is the principle of electroporation?

Answer 3

Electroporation is frequently used to transform bacteria, yeast, or plant protoplast by introducing foreign DNA. It is a very effecient method for transfection. The technique involves applying an electrical field to cells in order to increase the permeability of the cell membrane. The electrical pulse will make temporarily pores in the membrane, in which the nucleic acid can pass. After the electroporation, the cell membrane recovers, and the pores are closed.
The general steps in electroporation is:
(1) prepare the cells
(2) apply the electric pulse
(3) return the cells to growth conditions
(4) analyse the cells

Question 4

Why did we use this transformation method (electroporation)?

Answer 4

Electroporation is relatively easy to perform, can work well for cell types that are difficult to transfect, the method has reproducible result, no vector requirements, is less dependent of cell type, and has rapid transfection of large number of cells.

Question 5

What methods do we use to identify positive transformants and mutant cells?

Answer 5

The first step to identify positive transformants and mutated cells was to look for growth on the plates and in the flasks. Growth indicated that the cells are immune to the antibiotic, which is due to the ampicillin resistance gene in the plasmid.

Further, we performed PCR and HRM analysis.

Question 6

What is the advantages / disadvantages of this transformation technique (electroporation) compared other methods?

Answer 6

Advantages: Electroporation is relatively easy to perform, can work well for cell types that are difficult to transfect, the method has reproducible result, no vector requirements, is less dependent of cell type, and has rapid transfection of large number of cells.

Disadvantages: Requires special instruments, parameters must be carefully optimized, and potential for high toxicity as well as cell damage and mortality.

Question 7

What do we check for when we sequence the genome edited region? What can DNA sequence chromatograms tell us?

Answer 7

First of all we check if the region is successfully cloned into the organism. Then we check for any mutations in the genome edited region.

A four-color chromatogram displays the results of a sequencing run, where each of the nitrogenous bases is indicated by a specific color. So the results tells us the interpreted nucleotide sequence from 5´ to 3´.

A = green
C = blue
G = black
T = red

Question 7

What are the principles of CRISPR/Cas9?

Answer 7

CRISPR = clustered regularly interspaced short palindromic repeats.
A single gRNA, consisting of a crRNA sequence that is specific to the DNA target, and a tracrRNA sequence that interacts with the Cas9 protein, binds to a recombinant form of a Cas9 protein that has DNA endonuclease activity. The resulting complex will cause target-specific double-stranded DNA cleavage. The cleavage site will be repaired by the nonhomologous end joining (NHEJ) DNA repair pathway, an error-prone process that may result in INDELs that may disrupt gene function.

Question 8

How can we prove that a gene has been edited? What techniques did we use and what are the principles for these methods/ techniques?

Answer 8

HRM, Sanger sequencing, analysis of DNA sequence chromatograms.

Question 9

Are there any disadvantages or limitations with the CRISPR/Cas9 method?

Answer 9

The molecular mechanism exploited to insert DNA fragments is mediated by DNA repair machinery activated by the double strand break introduced by Cas9. Since the scope of the DNA repair system is not to integrate DNA fragments in the genome, targeted alleles often carry additional modifications such as deletions, partial or multiple integrations of the targeting vector, and even duplications.
(Off-target effects).
Delivery limitations.
Efficiency limitations.

Question 10

Describe the vector used in the lab experiment (transformation) and explain the function of its individual parts (promoters, terminators, selection markers, and expressed genes).

Answer 10

Promoters and terminators are stretches of DNA upstream and downstream of genes that control both the rate at which the gene is transcribed and the rate at which mRNA is degraded. Promoter is the region where the RNA polymerase binds to initiate transcription. A terminator is a sequence that causes the RNA polymerase to terminate transcription.
A selection marker is a gene introduced into cells which confers one or more traits suitable for artificial selection. An example of a selection marker is the Beta-lactamse, which confers ampicillin resistance.

Question 10

How does the Golden Gate assembly process work?

Answer 10

The principle consists of using a type IIS restriction enzyme and ligase in a restriction-ligation to assemble several DNA fragments in a defined linear order in a vector in a single step.
(1) BsaI flankes all DNA fragments, which makes an inward orientation
(2) BsaI flankes all plasmids, which makes an outward orientation
(3) Ligate

Question 11

Why do we use Type IIS restriction enzymes?

Answer 11

Type IIS restriction enzymes cleave the DNA outside of their DNA recognition site sequence. The restriction site of Bsal (GGTCTC / N1N2N3N4) consists of a DNA recognition site sequence (GGTCTC) and a DNA cleavage site (/) leading to a 4-nt single-stranded overhang after digestion. Because the expected circular recombinant DNA molecule does not contain restriction sites for the enzyme used, it cannot be re-digested, allowing restriction and ligation to be performed in the same reaction mix.

Question 12

How was Golden Gate assembly used to make a GFP tagged version of Thessus1 using the GreenGate system?

Answer 12

For GreenGate we choose Bsal.
The second important choice was the number of different modules which could be combined in a simple construct. Since the final plasmid only can be ligated when the DNA fragments representing all the modules is present, “dummy sequences” has to be introduced if certain modules isn`t necessary in a given construct. In the GreenGate system there are six modules which represents (1) the plant promotor, (2) an N-terminal tag, (3) the coding sequence of the gen of interest, (4) a C-terminal tag, (5) the plant terminator, and (6) the plant resistance cassette.
To make a GFP tagged version of Thessus1, we introduce plasmid consisting the GFP-gene.

Question 13

How do we transform E. coli with plasmid vectors?

Answer 13

E.coli is treated with CaCl2. Then we plate the transformed bacteria on plates containing LB medium with spectinomycin antibiotic and grow them overnight.

Question 14

How do you isolate plasmid DNA?

Answer 14

To isolate plasmid DNA we do a miniprep.
During the miniprep we first spin down the bacterial culture and discard the supernatant. The pellet in then resuspended in resuspension buffer. Then the lysis buffer is added. The solution is then incubated at room temperature for 1 minute. Then the neutralization buffer is added, and the tube is centrifuged. The supernatant is transferred to a spin column, and centrifuged. Plasmid wash buffer 1 is added, and centrifuged. Plasmid wash buffer 2 is added, and centrifuged. Sterile MQ water is added, the column in incubated, and then centrifuged. The plasmids will then be in the solution in the tube.

Question 14

How can we know that the plant plasmid vector is correctly assembled?

Answer 14

We perform a colony PCR.

Question 14

What is Sanger sequencing and how does that work?

Answer 14

Sanger sequencing is a method of DNA sequencing that involves electrophoresis and is based on the random incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication.

Question 15

What are the advantages with the Golden Gate assembly system?

Answer 15

It allows for the efficint and precise assembly of multiple DNA fragments into single construct. It can be used to create complex DNA constructs.

Question 16

How did we sterilize seeds and grow Arabidopsis plants in-vitro?

Answer 16

The seeds are sterilized in 70% ethanol, 50% bleach and then in autoclaved water for several times. The seeds are stored in a cold room overnight to stratify them, and then they are put in a growth cabinet for 6 days.
Stratified seeds are more likely to produce healthy and vigorous seedlings. These young plants are better equipped to compete with weeds, resist pests, and adapt to environmental conditions.

Question 17

What are the cellular effects of sorbitol on plants?

Answer 17

Sorbitol - which is a sugar alcohol - act as osmo-protectants, protective agents for cell membranes and as signaling molecules.
Sorbitol cause an osmotic stress that results in intensive production of intracellular glycerol, and submits the plant to controlled negative water potentials.

Question 18

How does BODIPY-based fluorescent molecular rotors operate? How can they be used to study viscosity?

Question 19

How can you perform time correlated single photon counting?

Answer 19

TCSPC records the time between an excitation pulse and a single emitted photon. A detector converts a single photon to an electronic pulse and the TCSPC electronics accurately measure its arrival time with respect to the excitation pulse.
The process may be thought of like a stopwatch, each emitted sample photon has its arrival time noted.

Question 20

What is the principle of Fluorescence Lifetime Imaging Microscopy (FLIM)?

Answer 20

FLIM exploits fluorescence lifetime which is a measure of how long a fluorescent molecule or fluorophore remains on average in its excited state before returning to the ground state by emitting a fluorescence photon.
FLIM-based detection is required in order to perform quantitative measurements of viscosity that are not biased by the variations in the concentration of the rotor, which cannot be easily determined in a heterogeneous microscopic environment.

Question 21

What is the function of the THESSUS1 kinase? How can THE1 affect cell wall integrity and plasma membrane viscosity?

Answer 21

THESSUS1 is a receptor-like kinase in plants that senses cell wall integrity and is active during cell wall perturbations.
The THE1-4 mutant is a gain of function mutant and has an increased expression of the gene. The the1-4 gene encodes for a THE1 protein with an absent kinase domain. This truncated protein induces stress responses more efficiently as the kinase domain is important for repressing the THE1 activity.

Question 22

What is pair-end sequencing?

Answer 22

Single-read sequencing involves sequencing DNA from only one end, and is the simplest way of utilize Illumina sequencing. Unlike single-read sequencing, paired-end sequencing allows users to sequence both ends of a fragment and generate high-quality, alignable sequence data. Paired-end sequencing facilitates detection of genomic rearrangements and repetitive sequence elements, as well as gene fusions and novel transcripts.
In addition to producing twice the number of reads for the same time and effort in library preparation, sequences aligned as read pairs enable more accurate read alignment and the ability to detect indel variants, which is more difficult with single-read data.

Question 23

What is indexing and why is that often used in RNAseq experiments?

Answer 23

Indexed sequencing is a method that allows multiple libraries to be pooled and sequenced together.

Question 24

Why can read depth be important for the analysis?

Answer 24

Read depth is the number of times a particular base is represented within all the reads from sequencing. The higher the read depth, the more confidence scientists can have in identifying a base - known as “base calling”.

Question 25

What is the difference between fasta and fastq?

Answer 25

FASTA stores a variable number of sequence records, and for each record it stores the sequence itself, and a sequence ID. In the context of nucleotide sequences, FASTA is mostly used to store reference data; that is, data extracted from a curated database.

FASTQ was conceived to solve a specific problem arising during sequencing: due to how different sequencing technologies work, the confidence in each base call varies. This is expressed in the Phred quality score. FASTA had no standardised way of encoding this. By contrast, a FASTQ record contains a sequence of quality scores for each nucleotide. FASTQ files are mostly used to store short-read data from high-throughput sequencing experiments.

Question 26

The edgeR package, what type of analysis can it perform?

Answer 26

edgeR is concerned with differential expression analysis rather than with the quantification of expression levels. It is concerned with relative changes in expression levels between conditions, but not directly with estimating absolute expression levels.

Question 27

How does prime editing work?

Answer 27

Prime editing is a gene editing method that can perform targeted small insertions, deletions, and base swapping in a precise way. The ability to delete bases is a hallmark of knockouts, while the ability to insert specific nucleotides in a precise manner is the premise underlying knock-ins. Prime editing enables targeted editing without generating double-stranded DNA breaks.
Prime editing is an all-in-one solution with small improvements in the CRISPR process that impact the final result - a classic sum-is-larger-than-the-parts scenario.

Question 27

What is a Cas9 nickase and how is it used in gene editing?

Answer 27

A Cas9 nickase in an engineered Cas9 that reduce potential off-target mutagenesis by inducing single-strand nicks rather than double-strand breaks.
When Cas9 nickase is nicking of the target strand, it stimulates cellular repair mechanisms, which leads to increased editing frequencies by both cytosine and adenosine base editors in the absence of a DSB.

Question 28

What do we mean by a “dead” Cas9 (dCas9) and what applications can it be used for?

Answer 28

Cas proteins devoid of nucleolytic activity (dead Cas proteins) can be used to deliver functional cargo to programmed sites in the genome.

Question 29

What is optogenetic gene editing?

Answer 29

Optogenetics uses genetically encoded tools, such as microbial opsins, to control cellular activities using light.
Optogenetic gene expression systems can control transcription with spatial and temporal detail unequaled with traditional inducible promoter systems.

Question 30

Does Cas nucleases only target dsDNA?

Answer 30

Cas proteins bind to target DNA or RNA and are guided into an activated state by crRNA, which can cleave not only specific DNA or RNA but also any dsDNA, ssDNA or ssRNA in the environment.

Question 31

Why can off-target effects be a concern and what can be done to reduce them?

Answer 31

Off-target effects are unintended consequences in an experiment. For CRISPR work, this often means that DNA cleavage at unanticipated sites.

There are several strategies to lower the predicted frequency of off-target events, many of which can be combined to lower them even further.
(1) Optimal gRNA selection
(2) Optimal Cas selection
(3) 2 gRNA approach

Question 32

What is a gene drive? Can the CRISPR/Cas9 method be used here?

Answer 32

A gene drive is a natural process and technology of genetic engineering that propagates a particular suite of genes throughout a population by altering the probability that a specific allele will be transmitted to offspring (instead of the Mendelian 50% probability).

Typically, scientists insert the gene drive into an organism´s DNA along with the CRISPR-Cas9 machinery. When the modified organism mates and its DNA mixes with that of its mate, the CRISPR-Cas9 tool cleaves the partner´s DNA at the same spot where the gene drive is located in the first organism. The cell repairs the cut DNA by copying the gene drive from the first organism into the corresponding spot in the DNA of the offspring. This means that both copies of the gene (one from each parent) now contain the gene drive.

Question 33

What are some of the principles behind light microscopy /fluorescence
microscopy / confocal microscopy?

Answer 33

In light microscopy, illuminating light is passed through the sample as uniformly as possible over the field of view.
In fluorescence microscopy, any dye molecules in the field of view will be stimulated, including those in out-of-focus planes.
Confocal microscopy provides a means of rejecting the out-of-focus light from the detector such that is does not contribute blur to the images being collected. This technique allows for high-resolution imaging in thick tissues.

Question 34

How can STED microscopy increase the resolution?

Answer 34

STED employs spatially modulated and saturable transitions between two molecular states to engineer improvements to the point-spread function. In STED microscopy, the specimen is illuminated by two synchronized ultrafast co-linear sources consisting of an excitation laser pulse followed by a red-shifted depletion laser pulse that is referred to as the STED beam. The effective resolution increase is completely dictated by the experimental configuration and the laser powers applied to the specimen.

Question 35

What is FRAP?

Answer 35

Fluorescence recovery after photobleaching (FRAP) is a method for determining the kinetics of diffusion through tissue or cells. It is capable of quantifying the two-dimensional lateral diffusion of a molecularly thin film containing fluorescently labeled probes, or to examine single cells. This technique is very useful in biological studies of cell membrane diffusion and protein binding.

Question 35

What is FRET?

Answer 35

Fluorescence resonance energy transfer (FRET) is a distance-dependent physical process by which energy is transferred nonradiatively from an excited molecular fluorophore (the donor) to another fluorophore (the acceptor) by means of intermolecular long-range dipole-dipole coupling. FRET can be an accurate measurement of molecular proximity at angstrom distances and highly efficient if the donor and acceptor are positioned within the Förster radius (the distance at which half the excitation energy of the donor is transferred to the acceptor).

Question 36

What are the common methods used to detect and identify proteins?