Introduction to practical course and seminar series · • In groups of two, each student will prepare a 30 min seminar on a topic related to ´Genetic Manipulation´ ... • Advantages,

Introduction to practical course and seminar series

Titel der PräsentationStruktureinheit der TU Dresden / Name Vorname des VortragendenOrt oder Anlass des Vortrags // 13.01.2018

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Student Seminars

• In groups of two, each student will prepare a 30 min seminar on a topic related to ´Genetic Manipulation´

• The topic will be selected two weeks prior to the seminar

• You will be given a review article on a given topic as a starting point to prepare the presentation

• You are then asked to select an original research article closely related to the review article • Article should not be more than 2 years old• Please email me your selected article by the first Friday after having recieved your seminar topic


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Grading of the Practical Course

• 30% Seminar presentation• 70% Practical course

• Participation• Lab protocol

30 min


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Presentation format

• Introduction to the topic and research article (~15min)• Theoretical background to the method• Advantages, disadvantages

• Brief introduction to research topic of chosen article• Why is this topic interesting?

• Presentation of results• You do not have to present all of the results!• It is better to carefully explain the key/most important results than gloss

over every result

• Conclusions and future directions

• Critique


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Tips on how to give a good presentation

You must NOT lose the audience

YOU are the focus, your slides are support

Confusing Boring


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• Know your audience• are they expert researchers in your field? Undergraduates? Non-scientists?

• Tell audience up front why they should care about the topic

• Tell a Story!• The transition between each slide should be very smooth• Avoid quantum leaps!

• Convey your excitement

• Keep it simple (less is more!!)

• Limit words – use visuals!• Avoid Death by PowerPoint: That dreaded presentation where the presenter reads off slide after slide after

slide after slide………………..• Use images and a few words when needed

https://www.elsevier.com/connect/how-to-give-a-dynamic-scientific-presentationhttp://blogs.nature.com/naturejobs/2017/01/11/scientific-presentations-a-cheat-sheet/

https://www.elsevier.com/connect/how-to-give-a-dynamic-scientific-presentation


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Choose an easy read FontSans serif vs Serif• Serifs are harder to read

Check Spelling!!


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Use the right amount of data on each slide

What you think they look like… What they actually look like…


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Blahblahblah…….

The outcome of these studies has demonstrated that sdkfjfjsdgkjsködghsdkhadkfhjsfjhksdv,akefhskfjhslEZTHLCKNSölektzslrtlowurlkfwrizsdlknskethwoöelgfknweotizuwoögjfweotuorgjroitjeotjgkjwepäouzpogkwöeltjwpojwpegojfwpojtopjfwejotwtjöfejwtejleöhtiöldvknwoiöhtadvfknwäetoha#vmäp6out#apkvpeotupfkwpotuüapfkpwetiüpkfvwpeofjaowihfhhwefewptojgeqirogjugmerpuotäpfjwakdfhakfhskdfhsölfkhfdlkjsälöuotsnkslgkhlfgksÖLEGIÄÖSDVLMÖSlrgjöSLDEOLEIWPaölejwpojtsdlömwöroztjsägjndärhjgmroutjmvsltkjlgfjs-lfksörjtösegmsdfmsdgsöljgölgjwgäwIORHGöwoervnbwoeröuwerwperowüprowüp

Do not overload your slides!!!!You MUST discuss every image on your

slide!!!


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Title each slide with its own premise


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Engage with the audience!

• Make eye contact with your audience!• Try and build up ‚drama‘ during the presentation – make the audience

want to hear what is coming on the next slides


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• A copy of this paper has been uploaded onto SELMA


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Seminar Schedule

Date Topic Presenter 1 Presenter 2

Nov. 18th Protein tagging (for localization) Anastasia Misco HaNgoc Anh

Nguyen

Nov. 18th Homologous recombination Lisa Göbner Katharina Hilbig

Nov. 25th Carla Hofmann Jannatul Rafeya

Nov. 25th Christoph Lux Paula Schröder

Dec. 9th Sandy Lemm Max BottPilar Lörzniig

Dec. 9th

Introduction to the Practical Course


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• Restriction Enzyme Digestion

• Gel Electrophoresis

• DNA cloning

• DNA amplification using PCR

• Genomic and cDNA libraries

• DNA sequencing

• Genome Expression

• Genetic Manipulation

• Recombinant protein expression

Techniques in Molecular Biology

Transcription: From DNA to RNA


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Central Dogma of Molecular Genetics


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Control of Gene Expression

• Different cell types synthesize different sets of proteins

2D-SDS PAGE of proteins expressed in two different cell types RED: common to both samplesBLUE: specific to one of the two samples


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• The same cell type can express a different set of proteins under different environmental conditions

Identification of highly nitrate-sensitive (HNS) gene set. a Transcriptome from the Nshort experiment. Heatmap shows average standardized transcript abundance for all genes in each response type (RT, n = 200, RT3 omitted).

Smith, S. et al (2019) Nature communication 10:4552


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(Splicing)

• For most genes transcriptional controls are most critical• How does a cell determine which of its thousands of

genes to transcribe?

Transcription: RNA polymerases


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RNA polymerase

• Transcription is catalyzed by the enzyme RNA polymerase

• Catalyzes the formation of a phosphodiester bond

Vorführender

Präsentationsnotizen

Catalyzes the formation of the phosphodiester bond Brings the complementary nucleoside-5’triphosphate onto the DNA template The 3’ OH group acts as a nucleophile attacking the innermost phosphate phosphorous atom of the incoming nucleoside triphosphate A pyrophosphate is release in the process


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RNA Polymerase

• RNA polymerase:• reads in the 3’- to 5’ direction and builds in the 5’ to 3’ direction• does not require a primers to initiate the process• cannot correct any mistakes made

How does the RNA polymerase know when to start transcription?


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Transcription

• RNA polymerase must be able to detect and transcribe specific segments of DNA• How does it do this??

Promoter site

• DNA contains regions of nucleotides called promoters that direct RNA polymerase to begin transcription

• These are regions of DNA that the RNA polymerase binds to very strongly

• there are two sequences that commonly act as promoters

TTGACA Bacterial GeneTATAATDNA Template5’

Pribnow Box(-10)(-35)

Bacterial promoters


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Consensus sequence for the major class of E. coli promoters

Probability of occurrence of each nucleotide in E. coli


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Eukaryotic Promoters

GGNCAATCT Eu GeneTATAA5’

(-25)(-75)

Enhancer

1. TATA box : found ~25 nucleotides upstream of transcription2. CAAT box: found ~75 nucleotides upstream3. Enhancer sequence: found to the right or left of start site


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Termination sites

• RNA polymerase will continue transcription until it reaches a termination sequence

• This termination sequence encodes for a terminal signal such as a hair-pin structure on the newly synthesized RNA molecule

Gene of Interest3’ Termination

RNA polymerase will spontaneously dissociate

How does the cell control the process of transcription?


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Gene Regulation - Prokaryotes

Prokaryotes can regulate gene expression by using special DNA binding proteins called repressors and activators

Repressor: blocks transcription

• If an inducer molecule is present that initiates gene expression then it can interact with the repressor gene and detach it from the operator

Activator (transcription factor): increases gene expression

• Most activators function by binding to specific DNA sequences


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The Bacterial Tryptophan Repressor

• The five enzymes that encode for tryptophan synthesis are arrange on a single operon

• A single promoter drives mRNA expression of all five genes


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The Bacterial Tryptophan Repressor

• Within the promoter is the gene regulatory element – operator

• The operator is recognised by the tryptophan repressor protein

Vorführender


When repressor occupies the operator it blocks the RNA polymerase access to the promoter sequence thereby preventing expression of the operon. When two moleculaes of tryptophan are bound to the repressor it is bound tothe operon


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Gene Regulation - Eukaryotes

TATAEnhancer

Core Promoter (TATA Box)• Over 50 proteins can bind to the core promoter• important complex for gene expression

Upstream Promoter (purple boxes)• Regions that can bind activator or repressor proteins needed to regulate

gene expression• Unlike the core promoters, these vary from gene to gene

Eu Gene


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Eukaryotic RNA polymerase


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Gene Regulation - Eukaryotes

TATAEnhancer

Enhancer• DNA segments typically located far away from the gene (upstream or

downstream)• Bind special transcription factors that increase the rate of transcription• Once the transcription protein binds onto the enhancer, the enhancer can

loop around and bind to the promoter region

Eu Gene

Practical Course

https://cloudstore.zih.tu-dresden.de/index.php/s/kJfR8xB7HL8xQgc


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• Restriction Enzyme Digestion

• Gel Electrophoresis

• DNA cloning

• DNA amplification using PCR

• Genomic and cDNA libraries

• DNA sequencing

• Genome Expression

• Genetic Manipulation

• Recombinant protein expression

Techniques in Molecular Biology

Diatoms


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Drosophila melanogaster

Saccharomyces cerevisiaeCaenorhabditis elegans

Zebrafish

Arabidopsis thaliana

Zea maysMouse


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Axlotl

Volvox

Naegleria gruberi

Oxytricha

Vorführender


Volvox: revealing the origins of multicellularity and germ–soma division of labor Naegleria gruberi: one cell with two extreme forms of motility Life with 16,000 chromosomes: Oxytricha as a model system to study genome biology, epigenetic inheritance, and somatic differentiation


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Diatoms• Unicellular photosynthetic microalgae• Cell wall made of silica• >100,000 species• Freshwater and marine species• Two main classes based on cell

symmetry• Centric (radial symmetry)• Pennate (bilateral symmetry)


Folie 4384th plate from Ernst Haeckl's Kunstformen der Natur (1904)


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Which biomolecules enable

SiO2 morphogenesis in diatoms ?


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http://oceancolor.gsfc.nasa.gov/SeaWiFS/

Diatoms are estimated to be responsible for ~20% of total

global carbon fixation


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PHYTOPLANKTON ZOOPLANKTON

PHOTOSYNTHESIS

CO2 O2

Light

Marine Food web

Vorführender


70% of earths surface is covered in oceans

Diatom evolutionary history


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Plant Evolutionary History

Proterozoic Paleozoic Mesozoic Cen

2500 542 252 65 0 (Mya)

Diatoms

Green algae

Land plants

1200

Red algae


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Why are diatoms so successful?

• Silica cell walls are:• energetically efficient to produce • a highly robust defense against predators• unlike the carbonate biominerals of other species are not as sensitive to

ocean pH• They have a very efficient way to dissipate excess solar energy, known as

non-photochemical quenching.• They are highly adaptive to different environments


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BLOOM and BUST

• their ability to outcompete all other species as soon as a nutrient limitation is removed

• In spring-summer, increased light and plentiful nutrients cause the diatoms to undergo a feeding frenzy.

• When they run out of nutrients, they enter the ‘bust’ phase of their life cycle, dying and sinking towards the bottom of the ocean.

• On their way down to the seafloor, they start to decompose at around 200 to 400 meters below the sea level, releasing the zinc and silicon back into the seawater.

Practical Course


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Nitrate uptake and assimilation in diatoms

NO3-

NH4+

NO2-

• Primary production in the ocean is largely regulated by the rate and magnitude of nitrate uptake and assimilation by microbes

• Within the marine phytoplankton the diatoms are the best competitors for high levels of NO3-

• Therefore in regions of high nutrient supply, diatom often domainte phytoplantkon communities


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Nitrogen Metabolism – KEGG Pathway


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Nitrogen uptake and assmilation in diatoms

NO3-

NH4+

NO2-

Nitrate reducatase(NR)

Nitrite reducatase(NiR)

UPTAKEASSIMILATION

NR enzyme is thought to be central to competitive

advantage of diatoms in the marine environment

Vorführender


Carbon and nitrogen metabolism are intricately linked in all organisms and are tightly regulated to maintain growth, homeostasis, and other cellular activities. In plants and algae, photosynthesis provides both carbon skeletons and the reductant needed for assimilation of inorganic NO3¯ by nitrate reductase (NR). NR catalyzes the conversion of nitrate (NO3¯) to nitrite (NO2¯), which can be further reduced to NH4+ for incorporation into amino acids and other biological molecules. In addition to being potential sources of biofuels and other high value products, marine algae are responsible for as much as 20% of global primary production.


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Urea Cycle

• is a metabolic pathway used in mammals to incorporate excess nitrogen into urea and remove it from the body.

• enables the diatoms to efficiently use carbon and nitrogen from their environment and could be a reason for the domination of diatoms in marine environments

• knockdown studies of the urea cycle enzymes demonstrated that the cycle enables diatoms to recover quickly after prolonged periods of nitrogen limitation.

Nature (2011) 473, 203–207).


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NR expression and localization is regulated by environmental conditions

McCarthy, J. et al (2017) The Plant Cell 29:2047-2070

How can we determine the DNA regulatory sequences (promoter & terminator) of the nitrate reductase gene?


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Green Fluorescent Protein (GFP)

Aequorea victoriaGreen Fluorescent Protein (GFP)

Live Cell imaging of fluorescently tagged proteins

http://www.tsienlab.ucsd.edu; http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2008/illpres.html


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GFP in Diatoms


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Using GFP to determine regulatory DNA sequences

• Promoter activity can be determined by cloning a promoter sequence upstream of a reporter gene and assaying the reporter activity

5‘UTR 3‘UTR3‘UTR

ATG stopstop

Upstream region contains the „ON„ switch for your gene of interest

TATA box

5‘UTR3‘UTRTATA box GFP

Take the upstream region from the genomic DNA and fuse to GFP reporter gene


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NR expression is regulated by environmental conditions

N source• NR mRNA transcripts are:

• nitrate inducible • nitrogen starvation inducible• ammonium repressible

• NR protein expression• nitrate inducible• nitrogen starvation repressible• ammonium repressible

Cylindrotheca fusiformis

N source

Poulsen, N. & Kröger, N. (2005) FEBS Journal 272: 3413-3423


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Using GFP to determine regulatory sequences


5‘UTRTATA box GFP

5‘UTRTATA box GFP

5‘UTRTATA box GFP

Generate different

truncations of promoter

sequence


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5‘UTRTATA box GFP

5‘UTRTATA box GFP

5‘UTRTATA box GFP

3‘UTR

downstream region contains the terminator

3‘UTR

3‘UTR

3‘UTR


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3‘UTR

downstream region contains the terminator


Does the terminator also contain nitrogen responsive regulatory

elements?Use the NR promoter with a

different terminator

heterologous terminator

Documents

Introduction to practical course and seminar series · • In groups of two, each student will prepare a 30 min seminar on a topic related to ´Genetic Manipulation´ ... • Advantages,