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Module 2: Expression Engineering
20.109Lecture 2
October 16th, 2007
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Public Understanding?
http:// www.ardenmoore.com/pets/dna-diet.html
Image removed due to copyright restrictions.
From “The DNA Diet,” AKC Gazette, April 2006:
“Scientists are seeking the recipes for dog foods that can attack illness at the genetic level”
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Public Understanding?
Images removed due to copyright restrictions.
Website screenshots from http://www.thednadiet.com, advertising a service using DNA testing to develop a personal diet plan.
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Public Understanding?
Images removed due to copyright restrictions.
Photo of anti-GMO protesters from http://www.gmfreeireland.org
Cartoon of “mad scientist” character
Editorial cartoon about fast-track bill to allow use of genetically modified food ingredients without labeling
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Public Understanding?
Image removed due to copyright restrictions.
Cartoon of “mad scientist”character.
Cite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MITOpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].
Text removed due to copyright restrictions.Excerpt from "Lawmakers, Administration Clash over Science Unding." by Aliya Sternstein. National Journal's Technology Daily, February 14, 2007.http://www.govexec.com/story_page.cfm?articleid=36137&ref=rellink (accessed May 7, 2008)
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Expression Engineering ExperimentDay 1 Day 2 Day 3
Day 4Day 5Day 6
Image of glowing luciferasein a microcentrifuge tube, removed due to copyright restrictions.
RT
Figure by MIT OpenCourseWare.
Image credits: Day 1 - Courtesy of Arkitek Studios. Used with permission. Day 2 - Courtesy of The Exploratorium. © The Exploratorium, http://www.exploratorium.edu. Day 4 – Figure by MIT OpenCourseWare. Day 5 – Dr. Natalie Kuldell. Day 6 – Courtesy of NIH.
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Expression Engineering ExperimentLecture 1
• intro to cell culture• intro to gene exp’n/RNAi
Lecture 2• transfection• luciferase
Lecture 3• off-target/nonspecific RNAi
Lecture 4• Writing lecture
(Neal Lerner)Lecture 5
• measuring gene express’n
Lecture 6• microarray analysis
(Rebecca Fry)Lecture 7
• high throughput technologies or RNAi applications (no lab)
Lecture 8• review of your data
Cite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MITOpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].
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DNA on the move: nature’s ways
Figure by MIT OpenCourseWare.
Phage infects Occasional phagepackages host DNA
Hybrid phageinfects new cell
Host may incorporate foreign DNA
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DNA on the move: nature’s ways1. Viral transduction
http://blogs.nature.com/news/blog/2006/10/ancient_human_virus_resurrecte.html
Cite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MITOpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].
Text and image removed due to copyright restrictions.Excerpt from: Pearson, Helen. "Ancient Human Virus Resurrected."Nature (31 October 2006). DOI:10.1038/news061030-4.
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DNA on the move: nature’s ways
1. Viral transduction
2. Conjugation
Figure by MIT OpenCourseWare.
Mobile plasmid Pilus
Bacterium
Chromosome
Donor
Recipient
Relaxosome Transferosome
New donor New donor
A
B
C
D
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DNA on the move: nature’s ways1. Viral transduction
2. Conjugation
3. Transformation
Jonathan M. Solomon and Alan D. Grossman “Who's competent and when: regulation of natural genetic competence in bacteria” Trends in Genetics (1996) 12(4):150
Courtesy Elsevier, Inc., http://www.sciencedirect.com. Used with permission.
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DNA on the move: in the lab1. Viral transduction
2. Conjugation
3. Transformation
chemical electrical
Ca
Ca
P P
P P
Figure by MIT O
penCourseW
are.
Courtesy of Zephyris.
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Ca
Ca
P P
P P
Figure by MIT O
penCourseW
are.
DNA on the move: in the lab1. Viral transduction
2. Conjugation
3. Transformation
chemical electrical
microinjection biolistics
Courtesy of Zephyris.
Cite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MITOpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].
Image removed due to copyright restrictions.Image removed due to copyright restrictions.
See http://firstivf.net/laboratory_tour.htm.
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Nature Biotechnology (2007) 25: 1149 - 1157 Mechanisms and optimization of in vivo delivery of lipophilic siRNAs
DNA on the move: RNAi delivery
Courtesy of Markus Stoffel. Used with permission.
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Nature Biotechnology (2007) 25: 1149 - 1157 Mechanisms and optimization of in vivo delivery of lipophilic siRNAs
DNA on the move: RNAi delivery
Courtesy of Markus Stoffel. Used with permission.
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DNA on the move: RNAi delivery
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DNA on the move: RNAi delivery
Courtesy of Mary Ann Liebert, Inc. publishers. Used with permission.
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DNA on the move: transgenic animals
®© The Nobel Foundation. Courtesy of Nobel Foundation. Used with permission. Cite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MITOpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].
Photos of the laureates removed due to copyright restrictions.Mario R Capecchi, Sir Martin J. Evans, and Oliver Smithies.
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DNA on the move: transgenic animals
Blastocyst Donor
ES Cell Donor
®© The Nobel Foundation. Courtesy of Nobel Foundation. Used with permission.
Cite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MITOpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].
Photos of the laureates removed due to copyright restrictions.Mario R Capecchi, Sir Martin J. Evans, and Oliver Smithies.
Photo of mice removed due to copyright restrictions.
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DNA on the move: transgenic animals
®© The Nobel Foundation. Courtesy of Nobel Foundation. Used with permission. Cite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MITOpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].
Photos of the laureates removed due to copyright restrictions.Mario R Capecchi, Sir Martin J. Evans, and Oliver Smithies.
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®© The Nobel Foundation. Courtesy of Nobel Foundation. Used with permission.
Courtesy of genOway. Used with permission.
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Photo of mice removed due to copyright restrictions.
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DNA on the move: transgenic animals
Cite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MITOpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].
Photo of "Alba, the fluorescent bunny," removed due to copyright restrictions.
See http://www.ekac.org/gfpbunny.html#gfpbunnyanchor.
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Courtesy of NOAA.2007: Exploring the Inner Space of the Celebes SeaCourtesy of Bruce Marlin, http://www.cirrusimage.com.
Used with permission.
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400 nm
700 nm
Courtesy of NASA.
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Fluorescence
Courtesy of NOAA.2007: Exploring the Inner Space of the Celebes Sea
Courtesy of Steven Haddock. Used with permission.Source: Haddock, S.H.D., C. M. McDougall, J. F. Case.
http://lifesci.ucsb.edu/~biolum/ (created 1997; updated 2007; accessed Fall 2007).
e-
e-
e-
e-
e-e-
Ground state orbital
Excited state orbitals
Nucleus
AbsorptionFluorescence
Figure by MIT OpenCourseWare.
Cite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MITOpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].
"The Bioluminescence Web Page."
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http://www.lifesci.ucsb.edu/~biolum/chem/
Bioluminescence
Cite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MITOpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].
Courtesy of Bruce Marlin, http://www.cirrusimage.com.Used with permission.
Chemical reaction diagrams removed due to copyright restrictions.See Figures 1, 2, and 4 in: http://www.chemistryexplained.com/Ar-Bo/Bioluminescence.html
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firefly luciferase renilla luciferase GFP
Images generated in Cn3D (http://www.ncbi.nlm.nih.gov/Structure/CN3D/cn3d.shtml)
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BLAST 2 Sequences
Photinus pyralis luciferase mRNA
Renilla reniformis luciferase mRNA
http://www.ncbi.nlm.nih.gov/BLAST/bl2seq/wblast2.cgiCite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MITOpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].
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PromegapsiCHECK2
Bioluminescence in the lab
Courtesy of Promega Corporation. Used with permission.
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Two bioluminescent reactions
Promega online information, Dual-Glo luciferase assayCourtesy of Promega Corporation. Used with permission.
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PromegapsiCHECK2
Bioluminescence in the labSee “quenching” of rxn with purified luciferase proteins
Courtesy of Promega Corporation. Used with permission.
Courtesy of Promega Corporation. Used with permission.
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It’s not easy being green
Any chance I could trade inmy GFP for luciferase?
Photo of "Alba, the fluorescent bunny," removed due to copyright restrictions. See http://www.ekac.org/gfpbunny.html#gfpbunnyanchor.
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Summary
1. DNA on the move
2. Let there be light
RNAi delivery
transgenic animals
“Mad scientist” cartoons removed due to copyright restrictions.
Ca
Ca
P P
P P
Figure by MIT O
penCourseW
are.
Images removed due to copyright restrictions.
Image Credits: Jellyfish photo courtesy of NOAA. 2007: Exploring the Inner Space of the Celebes Sea.Firefly photo: Courtesy of Bruce Marlin, http://www.cirrusimage.com. Used with permission.psiCHECK-2 Vector plasmid map: Courtesy of Promega Corporation.
Cite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MITOpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].
Courtesy of Bruce Marlin,http://www.cirrusimage.com.Used with permission.
Courtesy of NOAA.2007: Exploring the Inner Space of the Celebes Sea