Difference between revisions of "20.109(S11):Complete DNA design (Day2)"
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==Introduction== | ==Introduction== | ||
− | + | The edge detector design harnesses a lot of interesting biology, such as 2-component signaling and quorum sensing. We'll talk about these ways that bacteria get information about the outside world next week. For today, our focus is on bacteriophage lambda, in order to best understand how we might modify the P<sub>lux-λ</sub> promoter. | |
− | also | + | Bacteriophage, or phage for short, are viruses that infect bacteria. They are abundant and also various in their physical characteristics and the mechanisms they use to propagate. Phage lambda is a very well-characterized phage and an excellent model for understanding the fundamentals of gene regulation, as described by Mark Ptashne in his excellent book ''A Genetic Switch: Phage λ and Higher Organisms''. Despite having only 50,000 bp of DNA to work with, λ is able to exist in two distinct states — lytic or lysogenic — and switches between them depending on environmental cues. The switch is effected through a sophisticated interplay of molecular interactions among proteins and DNA. |
− | + | While you do not need to understand the mechanism of the switch in detail, you do want to be familiar with most of its components. The switch comprises two promoters and three operators, of which one promoter and two operators are part of P<sub>lux-λ</sub>. The image below shows the DNA that makes up promoter P<sub>R</sub>. Notice that P<sub>R</sub> (bracketed) has some overlap with two different operator sites, O<sub>R</sub>1 and O<sub>R</sub>2 (inside green boxes). The -35 and -10 regions of the promoter are shown in boldface blue text; notice again that these overlap in part with the operator sites. The leftmost -35 region belongs to a different promoter, P<sub>RM</sub>, whose associated gene is transcribed in the opposite direction; this promoter also overlaps with the third of three operator sites, O<sub>R</sub>3, that together comprise operator O<sub>R</sub> (the R stands for "right"). One thing you want to understand about the operator sites is that they behave cooperatively. | |
− | + | [[Image:S11-M2_lambda-PR.jpg|thumb|center|600px|'''Phage λ promoters and operators, adapted from M. Ptashne.''' See text above for complete description. This image is adapted from Figure 2.16 in the second edition of ''A Genetic Switch''.]] | |
− | + | The promoter P<sub>R</sub> does not require a transcriptional activator to turn on. However, it can be repressed by a protein that should be familiar to you from last time, the lambda repressor that is encoded by the ''cI'' gene. Repressor has about a ten-fold higher affinity for O<sub>R</sub>1 than for O<sub>R</sub>2, but once the former is bound, the latter's affinity increases. In fact, the attractive physical interaction between two repressor proteins results in their nearly concurrent binding at the two operator sites. | |
− | + | By now, your reading about promoters (and operators) — both general and specific to λ and ''lux'' — should enable you to develop a modification to P<sub>lux-λ</sub> that reduces its leakiness and thereby improves the edge detector. | |
− | + | [[Image:Be109EcoRIsite.jpg|thumb|right|200px|'''EcoRI cuts between the G and the A on each strand of DNA, leaving a single stranded DNA overhang (also called a sticky end ) when the strands separate.''']] | |
− | + | As a practical matter, you also want to understand a bit about restriction enzymes today. Also called restriction endonucleases, these proteins cut ( digest ) DNA at specific sequences of bases. The restriction enzymes are named for the prokaryotic organism from which they were isolated. For example, the restriction endonuclease ''EcoRI'' (pronounced echo-are-one ) was originally isolated from ''E. coli'' giving it the |