Difference between revisions of "20.109(S11):Complete DNA design (Day2)"
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==Introduction== | ==Introduction== | ||
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+ | 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. | ||
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+ | 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. | ||
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+ | 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. | ||
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+ | [[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''.]] | ||
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+ | 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. | ||
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+ | 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. | ||
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+ | [[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.''']] | ||
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+ | 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 Eco part of the name. RI indicates the particular version on the ''E. coli strain'' (RY13) and the fact that it was the first restriction enzyme isolated from this strain. | ||
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+ | The sequence of DNA that is bound and cleaved by an endonuclease is called the recognition sequence or restriction site. These sequences are usually four or six base pairs long and palindromic, that is, they read the same 5 to 3 on the top and bottom strand of DNA. For example, the recognition sequence for EcoRI (see also figure at right) is | ||
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+ | <font face="courier"> | ||
+ | 5 GAATTC 3 <br> | ||
+ | 3 CTTAAG 5 | ||
+ | </font> | ||
+ | |||
+ | <br style="clear:both" /> | ||
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+ | [[Image:Be109HaeIIIcrystals.jpg|thumb|left|200px|'''HaeIII crystals''']] | ||
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+ | Unlike EcoRI, some other restriction enzymes cut precisely in the middle of the palindromic DNA sequence, thus leaving no overhangs after digestion. The single-stranded overhangs resulting from DNA digestion by enzymes such as EcoRI are called sticky ends, while double-stranded ends resulting from digestion by enzymes such as HaeIII are called blunt ends. HaeIII recognizes | ||
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+ | <font face="courier"> | ||
+ | 5 GGCC 3 <br> | ||
+ | 3 CCGG 5 | ||
+ | </font> | ||
+ | |||
+ | <br style="clear:both" /> | ||
==Protocols== | ==Protocols== | ||
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===Part 1: Complete DNA design=== | ===Part 1: Complete DNA design=== | ||
− | + | For homework you were asked to think about what feature(s) of P<sub>lux-λ</sub> (at least the version of this hybrid promoter present in the edge detector) might be causing its suboptimal behavior. Now is your chance to fix it, by specifying mutations, deletions, and/or additions to the sequence. We have built a version of the IPTG-sensitive pseudo-edge detector with restriction sites directly bracketing P<sub>lux-λ</sub>, thus allowing the faulty part to be readily swapped out with a modified version. | |
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+ | You are welcome to make as subtle or drastic a change as you wish to reduce the promoter's leakiness. We hope to see a variety of solutions implemented by the class at large, so do post your ideas on the Talk page and consider changing your design if it is identical to another group's. | ||
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+ | You will implement your design by specifying two synthetic oligonucleotides — a top strand and a bottom strand — that can be annealed together and then ligated directly into a digested pED-IPTG-INS backbone. (Where ED indicates pseudo-edge detector, IPTG the molecule that it is sensitive to, and INS the fact that P<sub>lux-λ</sub> variants can readily be ''ins''erted into the vector.) | ||
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+ | #Begin by copying the P<sub>lux-λ</sub> sequence into a Word document. | ||
+ | #Underline the lambda operators and the lux box, and mark the -10 and -35 regions in bold. | ||
+ | #Make a copy of the annotated P<sub>lux-λ</sub> sequence below the original, and modify the sequence to reflect your design. Indicate which are the 5' and 3' ends. | ||
+ | #Now get the complement of this strand (for example, using [http://arep.med.harvard.edu/labgc/adnan/projects/Utilities/revcomp.html this website]) and mark its 5' and 3' ends as well. | ||
+ | #Within the backbone, the restriction sites bracketing P<sub>lux-λ</sub> are ''XmaI'' at the 5' end and ''BamHI'' at the 3' end. Add the appropriate restriction overhangs to each of your strands such that they will be ligated into your backbone without digestion. | ||
+ | #*It's perhaps easiest to check that you are doing this correctly by writing out a bit of the backbone sequence before and after digestion (by hand), doing the same for the insert, and making sure that they will fit together. | ||
+ | #*A great resource for information about restriction enzymes is the [http://www.neb.com/nebecomm/default.asp NEB website]. You can begin at at ''Technical Reference'' → ''Enzyme Finder''. | ||
+ | #For ordering purposes, DNA should always be written 5' to 3'. Add your two sequences to the Day 2 Talk page in this format, and also hand in a copy of your design document when you are done with it. Paste a second completed copy in your notebook. | ||
+ | #Write a 1-3 sentence description of your design rationale, both in the document and on the Talk page table. | ||
− | + | ===Part 2: Test liquid cultures for β-gal production=== | |
+ | With this assay you will determine the amount of beta-galactosidase activity associated with your cultures from last time. A table is included here to help you organize your assay, but you can make one of your own if you prefer. | ||
− | + | When you first try this assay you may find 15 second intervals too fast, and when you are expert at it you may find them too slow. Somewhere between 10 and 20 seconds should be a good time interval to shoot for. | |
− | The sample order below is recommended in order to minimize the risk of saturating samples expected to produce a lot of | + | The sample order below is recommended in order to minimize the risk of saturating samples expected to produce a lot of β-gal ("neg" means no additives). '''When you first try this assay you are almost certain to incubate some samples too long. Remember that you are shooting for a subtle rather than a bright yellow.''' |
<center> | <center> | ||
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</center> | </center> | ||
− | # Retrieve the cultures that you prepared last time, and prepare | + | # Retrieve the cultures that you prepared last time, and prepare 1 mL of a 1:10 dilution of each one in Zbuffer. |
#*Use 0.6 mL of the dilution to measure the OD<sub>600</sub> for each sample. | #*Use 0.6 mL of the dilution to measure the OD<sub>600</sub> for each sample. | ||
− | #*Save the rest for the | + | #*Save the rest for the β-gal assay. |
− | # Add | + | # Add 450 μl of Zbuffer to 9 eppendorf tubes labeled 0-8. |
− | # Add | + | # Add 50 μl of the diluted cells to each tube. See chart above for guidance. Add 50 ul of Zbuffer to tube 0, to serve as your blank. |
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# Next you will lyse the cells by add 20 μl of 0.1% SDS to each eppendorf. | # Next you will lyse the cells by add 20 μl of 0.1% SDS to each eppendorf. | ||
− | # To more fully lyse the cells, you should also add 30 μl of chloroform (CHCl<sub>3</sub>) to each tube. Do this in the hood since chloroform is volatile and toxic. #*You will need to hold the pipet tip close to the eppendorf as you move between the chloroform stock bottle and your eppendorfs since chloroform has a low surface tension and will drip from your pipetmen. Be sure to dispose of your pipet tips in the chloroform waste container located on the right side of the hood. | + | # To more fully lyse the cells, you should also add 30 μl of chloroform (CHCl<sub>3</sub>) to each tube. Do this in the hood since chloroform is volatile and toxic. |
+ | #*You will need to hold the pipet tip close to the eppendorf as you move between the chloroform stock bottle and your eppendorfs since chloroform has a low surface tension and will drip from your pipetmen. Be sure to dispose of your pipet tips in the chloroform waste container located on the right side of the hood. | ||
#To complete the cell lysis, vortex the tubes for 10 seconds each. You should time these precisely since you want the replicates to be treated as identically as possible. | #To complete the cell lysis, vortex the tubes for 10 seconds each. You should time these precisely since you want the replicates to be treated as identically as possible. | ||
#*You should be able to fit 2-3 tubes on the vortex at once. | #*You should be able to fit 2-3 tubes on the vortex at once. | ||
− | # Start the reactions by adding 100 μL of ONPG to each tube at 10 second intervals, including your blank. Invert to mix. | + | # Start the reactions by adding 100 μL of ONPG to each tube at 10 second intervals (or whatever Δ t you have chosen), including your blank. Invert to mix. |
# Stop the reactions by adding 250 μL of Na<sub>2</sub>CO<sub>3</sub> to each tube once sufficient yellow color has developed. | # Stop the reactions by adding 250 μL of Na<sub>2</sub>CO<sub>3</sub> to each tube once sufficient yellow color has developed. | ||
− | #* | + | #* |