Concept explainers
The researchers who investigated bioluminescence and quorum sensing found that E. coli transformed with a plasmid containing a 9 kb fragment of V.fischeri DNA could glow when the cell population was dense. They mutagenized these E. coli cells and isolated many mutations that mapped to the 9 kb fragment and prevented the cells from glowing. They then performed complementation testing on these mutants by transforming E. coli cells simultaneously with two plasmids, each containing the 9 kb fragment with one of the mutations. To ensure the E.coli cells were transformed with both plasmids, one of the two plasmids had a gene conferring resistance to ampicillin, while the other plasmid had a gene conferring resistance to tetracycline, and cells were selected on petri plates that had both antibiotics.
a. Construct a 9 × 9 complementation table for the nine mutations list that follows, using “+” to indicate cells that would glow and “-” to indicate cells that would remain dark. (You only need to fill in half the table.)
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To construct:
A 9
Introduction:
The researchers investigating bioluminescence and quorum sensing found out that the E.coli that is transformed with a plasmid containing a 9 kb fragment of Vibrio fischeri glows when the cell population becomes dense. Many of the E.coli cells were mutagenized and isolated that mapped to the 9 kb fragment and prevented the cells from glowing.
Explanation of Solution
The following table represents the 9
Mut1 | Mut2 | Mut3 | Mut4 | Mut5 | Mut6 | Mut7 | Mut8 | Mut9 | |
Mut1 | - | + | + | + | + | + | + | - | - |
Mut2 | - | + | + | + | + | + | - | - | |
Mut3 | - | + | + | + | + | - | - | ||
Mut4 | - | + | + | + | - | - | |||
Mut5 | - | + | - | + | + | ||||
Mut6 | - | - | + | + | |||||
Mut7 | - | + | + | ||||||
Mut8 | - | - | |||||||
Mut9 | - |
The genes that play an important role in the bioluminescence of Vibrio fischeri are luxR, luxICDABE, and luxI. luxR produces LuxR protein, which is a transcriptional activator required for the transcription of luxICDABE mRNA. luxICDABE involves a polycistronic region producing the proteins LuxI, LuxC, LuxD, LuxA, LuxB, and LuxE. The LuxI is a type of synthase enzyme that synthesizes an autoinducer. The autoinducer binds to LuxR protein and enables it to bind DNA. With reference to figure 1, the negative sign indicates that the combination of mutations does not complement each other; and due to this reason, the cells remain dark. The presence of positive sign indicates that the mutation complement each other, and the cells tend to glow.
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Chapter 15 Solutions
Genetics: From Genes to Genomes, 5th edition
- To determine if the antibiotic resistance in MH1 was carried on a plasmid, you first isolate the plasmid in MH1 using the plasmid DNA purification technique. Then, you transform bacteria that are not resistant to penicillin/ampicillin with the plasmid isolated from MH1. For the bacterial transformation experiment, you set up the three controls listed below. Match each control with its appropriate purpose (i.e. what it is controlling for) Please note: Transformed bacteria are bacteria that received the plasmid from MH1 and untransformed bacteria are bacteria that did not receive a plasmid. Testing to ensure that the bacteria used in the transformation experiment are viable (i.e. can grow on LB media) (Choose) [ Choose ) after transformation. Untransformed bacteria plated on LB only plate Testing to ensure that the bacteria used in the transformation experiment are viable (ie. can grow on LB media) before transformation. Transformed bacteria plated on LB only plate Untransformed bacteria…arrow_forwardWhen using a conventional plasmid cloning vector containing a b-galactosidase gene, it is possible to perform a "blue-white screen" to determine which bacteria have taken up a plasmid into which a DNA fragment as been inserted, as opposed to those that have taken up just reclosed plasmid vector, by growing the transformed cells on nutrient agar plates containing the artificial b-gal substrate X-gal. Will bacteria that have taken up a plasmid into which a DNA fragment has been inserted form a blue colony or a white colony when grown on this medium? Briefly explain why these bacteria would form a colony of the color you chose.arrow_forwardYou have set up a recombinant DNA experiment using the plasmid PBR322 as the vector (see plasmid below). You use the BamHI restriction site on the plasmid to insert the target DNA. The plasmid is then used to transform E.coli colls Is the following statement True or False? Growth of the transformed cells on agar containing both ampicillin and tetracycline will eliminate any cells that do not contain a plasmid. Clal Hindlll EcoRI Pvul BamHI Pstl amp tet PBR322 -Sall ori rop Pvull True Falsearrow_forward
- A plasmid that is both ampicillin and tetracyclineresistant is cleaved with PstI, which cleaves within theampicillin resistance gene. The cut plasmid is ligated withPstI-digested Drosophila DNA to prepare a genomic library,and the mixture is used to transform E. coli K12. Question: If recombinant cells were plated on medium containingampicillin or tetracycline and medium withboth antibiotics, on which plates would you expectto see growth of bacteria containing plasmids withDrosophila DNA inserts?arrow_forwardFor protein expression, a different strain of Coli is being employed than the one used for plasmid DNA expression. You disinfect your bench in advance of transforming the plasmid into the other cell line after completing the miniprep to isolate the plasmid. After the transformation is finished, you place the two samples in the freezer. You discover two separate tubes with the labels A and B when you try to get the cells for use. You decide to label your samples more accurately the following time. Since it costs a lot to buy competent cells, you design an experiment to find out what kind of cells are in each tube. Give an example of an experiment you could do to identify the cells using the equipment in our lab. Here, cloning is not an option. Small explanation with citation pleasearrow_forwardA plasmid that is both ampicillin and tetracyclineresistant is cleaved with PstI, which cleaves within theampicillin resistance gene. The cut plasmid is ligated withPstI-digested Drosophila DNA to prepare a genomic library,and the mixture is used to transform E. coli K12. Question: Which antibiotic should be added to the mediumto select cells that have incorporated a plasmid?arrow_forward
- Shown below is a diagram for a plasmid vector you want to use to clone a gene. The diagram shows the location of the recognition sites for four restrictions enzymes, BamHI (B), EdoRI (E), Hindill (H), and Xhol (X). The genes encoding beta-lactamase (AmpR) and beta-galactosidase (lacZ) are indicated. If you were to use this vector, which enzyme should be used to linearize the plasmid in preparation for cloning? E B lacz O Hindi!! BamHI O EcoRI O Xhol H EcoRI and Xhol E -X AmpRarrow_forwardA plasmid, pUC18, contains the ampicillin-resistance gene, the origin of replication, and the ß - gal gene, which codes for the B-galactosidase protein. This protein can break down the synthetic chemical X-gal, producing a blue product that stains the entire cell blue (but is harmless to the bacteria). At the beginning of the B-gal gene there are several unique restriction sites (some of them are shown in the diagram below). You wish to clone a 1.0-kb Xbal fragment into the pUC18 plasmid, so you cut the plasmid with Xbal and, after removing the enzyme, mix the Xbal-cut plasmid with the 1.0-kb fragment, ligate, and transform competent bacteria. Pati Xbal EcoRI B-gal A Amp ori Figure: pUC18 plasmid map (a) On what medium would you grow your transformed bacteria? (b) Do you expect the bacteria carrying plasmid pUC18 (without the insert) to be blue or white when grown in the presence of X-gal? Explain.arrow_forwardMany resistance mechanisms are encoded on plasmids. These mechanisms are of great clinical significance, because they can spread very easily through horizontal gene transfer. A culture of the bacterial isolate is grown, and plasmid DNA is isolated using a spin column-based solid phase extraction method. The purified plasmid DNA is then submitted for next-generation sequencing. Bioinformatic analyses of the sequencing results suggests that the following gene is likely involved in antibiotic resistance: > putative antibiotic resistance gene ATGCGTGTATTAGCCTTATCGGCTGTGTTTTTGGTGGCATCGATT ATCGGAATGCCTGCGGTAGCAAAGGAATGGCAAGAAAACAAAAGT TGGAATGCTCACTTTACTGAACATAAATCACAGGGCGTAGTTGTG CTCTGGAATGAGAATAAGCAGCAAGGATTTACCAATAATCTTAAA CGGGCGAACCAAGCATTTTTACCCGCATCTAGTGCGAAAATTCCC AATAGCTTGATCGCCCTCGATTTGGGCGTGGTTAAGGATGAACAC CAAGTCTTTAAGTGGGATGGACAGACGCGCGATATCGCCACTTGG AATCGCGATCATAATCTAATCACCGCGATGAAATATTCAGTTGTG CCTGTTTATCAAGAATTTGCCCGCCAAATTGGCGAGGCACGTATG…arrow_forward
- You have just carried out a transformation using a plasmid (possessing a Amp-resistance gene) that was 0.001 ug/ul in concentration. You added 10 ul of this plasmid to 100 ul of a bacterial cell suspension. Then you added 250 ul of LB following heat shock. After plating 200 ul of this cell suspension with LB onto a LBA plate, you observe 8 colonies on this plate. What is the transformation efficiency of this plasmid (in colonies/ug of plasmid) based on these results? about 1400 O about 80O about 4000 O about 400O O about 8000 f6 米 IO fs fg f1o 19 08. O OOarrow_forwardThe plasmids from the pUC series are created in the University of California. They carry a lacz gene that plays a significant role in the screening process after transformation. (i) Name the screening process utilizing the lacZ gene. (ii) Elaborate how this gene plays a crucial role in the screening step as mentioned above.arrow_forwardIn biotechnology, gene cloning is a crucial technique used in many genetic modification experiments. A vector is normally required to perform this process. The vector commonly used to transform a bacterial host cell is known as a plasmid. Below is the general plasmid map of pBR322. Elaborate the functions of ori, Ap and TeR present in pBR322. Bul 3759 Prul 3733 PM 3507 BDI 3602 Asel 3537 Bal 3433 Finci 3905 Scal 3844 3787 Bad 3420 Ahdi 3361 Acul 3000 AlwNI 2884 Sspl 4168 Earl 4155 Acul 4048 Xust 3961 Bell 2777 Bc1 2682 ori Aall-Zeal 4284 BeiVI 4209 B 4205 Del 2575 Pail-Att 2473 BAB 2404 EcoRI 4350 Earl 2351 BspQI-Sapl 2350 Ndel 2295 BAP 2291 178-Accl 2244 Cial-Ispit 23 Hindill 29 pBR322 4,361 bp Ball 2225 TthJ11- Part 2217 rop EcoRY 185 Bmtl-Nhel 229 Hamill 375 Sgr 400 Ball 471 Xml 2029 Pull 2064 Dalt 2162 BamB 2122 Banil 485 Bigl 128 Sphl 582 EcoN1 822 Sall-Accl-Hell 651 Pshu 712 BsaBl 1668 Engl 939 BeY1 945 Nrul 972 BAPT 1045 BspMI-BfuM 1063 PAMI 1315 Bumi 1353 Bgl 1650 BapEl 1664…arrow_forward
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