introduction to practical course and seminar series · • in groups of two, each student will...
TRANSCRIPT
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Introduction to practical course and seminar series
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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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Titel der PräsentationStruktureinheit der TU Dresden / Name Vorname des VortragendenOrt oder Anlass des Vortrags // 13.01.2018
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Grading of the Practical Course
• 30% Seminar presentation• 70% Practical course
• Participation• Lab protocol
30 min
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Titel der PräsentationStruktureinheit der TU Dresden / Name Vorname des VortragendenOrt oder Anlass des Vortrags // 13.01.2018
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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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Titel der PräsentationStruktureinheit der TU Dresden / Name Vorname des VortragendenOrt oder Anlass des Vortrags // 13.01.2018
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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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Titel der PräsentationStruktureinheit der TU Dresden / Name Vorname des VortragendenOrt oder Anlass des Vortrags // 13.01.2018
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Tips on how to give a good presentation
• 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/
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Titel der PräsentationStruktureinheit der TU Dresden / Name Vorname des VortragendenOrt oder Anlass des Vortrags // 13.01.2018
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Tips on how to give a good presentation
Choose an easy read FontSans serif vs Serif• Serifs are harder to read
Check Spelling!!
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Titel der PräsentationStruktureinheit der TU Dresden / Name Vorname des VortragendenOrt oder Anlass des Vortrags // 13.01.2018
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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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Titel der PräsentationStruktureinheit der TU Dresden / Name Vorname des VortragendenOrt oder Anlass des Vortrags // 13.01.2018
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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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Titel der PräsentationStruktureinheit der TU Dresden / Name Vorname des VortragendenOrt oder Anlass des Vortrags // 13.01.2018
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Tips on how to give a good presentation
Title each slide with its own premise
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Titel der PräsentationStruktureinheit der TU Dresden / Name Vorname des VortragendenOrt oder Anlass des Vortrags // 13.01.2018
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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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Titel der PräsentationStruktureinheit der TU Dresden / Name Vorname des VortragendenOrt oder Anlass des Vortrags // 13.01.2018
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• A copy of this paper has been uploaded onto SELMA
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Titel der PräsentationStruktureinheit der TU Dresden / Name Vorname des VortragendenOrt oder Anlass des Vortrags // 13.01.2018
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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
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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
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Transcription: From DNA to RNA
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Titel der PräsentationStruktureinheit der TU Dresden / Name Vorname des VortragendenOrt oder Anlass des Vortrags // 13.01.2018
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Central Dogma of Molecular Genetics
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Titel der PräsentationStruktureinheit der TU Dresden / Name Vorname des VortragendenOrt oder Anlass des Vortrags // 13.01.2018
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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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Titel der PräsentationStruktureinheit der TU Dresden / Name Vorname des VortragendenOrt oder Anlass des Vortrags // 13.01.2018
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Control of Gene Expression
• 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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Control of Gene Expression
(Splicing)
• For most genes transcriptional controls are most critical• How does a cell determine which of its thousands of
genes to transcribe?
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Transcription: RNA polymerases
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RNA polymerase
• Transcription is catalyzed by the enzyme RNA polymerase
• Catalyzes the formation of a phosphodiester bond
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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
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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
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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
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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
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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
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Diatoms
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Drosophila melanogaster
Saccharomyces cerevisiaeCaenorhabditis elegans
Zebrafish
Arabidopsis thaliana
Zea maysMouse
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Axlotl
Volvox
Naegleria gruberi
Oxytricha
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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)
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Folie 4384th plate from Ernst Haeckl's Kunstformen der Natur (1904)
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Titel der PräsentationStruktureinheit der TU Dresden / Name Vorname des VortragendenOrt oder Anlass des Vortrags // 13.01.2018
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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
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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.
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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
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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
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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‘UTR3‘UTRTATA box GFP
5‘UTRTATA box GFP
5‘UTRTATA box GFP
5‘UTRTATA box GFP
Generate different
truncations of promoter
sequence
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Using GFP to determine regulatory sequences
5‘UTR3‘UTRTATA box GFP
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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Using GFP to determine regulatory sequences
5‘UTR3‘UTRTATA box GFP
3‘UTR
downstream region contains the terminator
5‘UTR3‘UTRTATA box GFP
Does the terminator also contain nitrogen responsive regulatory
elements?Use the NR promoter with a
different terminator
heterologous terminator