Question

In 1974 Fred Sanger had managed to complete sequencing of Phage Phi x174. It was the first genome ever sequenced. Since then, scientist have successfully introduced and extensively taken advantage of genetic material allowing them to ask interesting questions and “shuffle” genes.

Your goal is now to play with bioluminescence and test the expression of the previously isolated bacterial Lux operon, Aliivibrio fischeri is a Gram-negative, rod-shaped marine bacterium with bioluminescent properties; it is a heterotrophic symbiote with various marine animals, including Hawaiian bobtail squid. The bacterial luciferin-luciferase system is encoded by a set of genes within the lux operon. In A. fischeri, five genes (luxCDABEG) are active in the emission of visible light, and two genes (luxR and luxI) are involved in operon regulation. Several external and intrinsic factors can induce or inhibit the transcription of this operon and produce or suppress bioluminescence.
Note that for your experiment, the well-conserved inducible promoter IS PART of the Lux operon with all 5 critical genes you need to produce bioluminescence, while both luxI and LuxR genes ARE NOT part of your study; hence, your simple task is to express this complete 5-gene operon and quantify the phenotype. Design your experiment, if you are starting with the relatively low concentration of template DNA coding for lux Operon, including the promoter. The staring DNA concentration is “clean” (260/280 is 2.0, and you already treated your sample with RNAse and DNAse I), but not sufficient to proceed with cloning.

Provide a flow-chart of your experimental design and DRAW and DISCUSS ALL “MUST-HAVE” recombinant bacteriophage characteristics you need to perform your experiment. Include the choice of your restriction enzymes and show their place with the vector (blunt vs. sticky argument) and discuss (+) and (-) controls. Importantly, you must be able to observe and quantify the promoter / operon activity of your system, so propose how to “capture the signal” in vitro. Moreover, since the budget allows, you insist on confirming the integrity of your recombinant plasmid by periodically isolating a single-stranded copy to be sent for sequencing and ultimately tagging the critical protein of interest with the antibody in case you move to “in vivo” phase of the study to verify cellular compartment expression within the bacterial host.

Answer 1

Lux genes codes for reporter and enzymes required for production of its substrate( decanal )

1. Genomic DNA extraction by Phenol-chloroform, Primer designing using nucleotide sequence of lux genes from sequence retrived from NCBI . PCR amplification was performed (pcr cycles 5 min at 94C , annhealing 58C, 60 C at Extension and final extension at 72C). PCR product allowed to run on agarose gel. Purification of PCR product by DNA extraction kit.

Sequencing of PCR product using Chain termination method.

2. Cloning of lux genes into cloning vector(plasmid)

PCR AMPLIFIED PRODUCT WAS LIGATED INTO 3'tailed Restriction digested plasmid vector, and tranformed into Ecoli DH5 COMPETENT CELLS.

PCR amplification of the lux A, B,C,D, G genes using primers to introduce the unique restriction sites at the ends(5' AND 3' ENDS).

Digestion of PCR amplified fragment with restriction enzymes, cloning at compatible sites downstream of Gal inducible promoter.

Ecoli DH5 were grown in LB Media, competent cells were prepared with required antibiotic concentration(e.g 100ug/ml) ampicillin,Cells are transformed with ligation mixture by electroporation in DH5, Selection of antibiotic resistant colonies.

Isolation of plasmid DNA from recombinant positive clones.

Sequencing of plasmid DNA to check orientation of the lux genes.

PROTEIN PURIFICATION

Cells were grown in 50 ml LB media with ampicillin overnight culture was induced with galactose , SINCE IT IS UNDER GAL inducible promoter. Cells were harvested, protein was extracted and allowed to run on SDS PAGE gel . Staining in coomasie followed by destained and then expression was compared in induced and uninduced samples.

Western blotting using antibody specific . GEL RUN -- TRANSFER ONTO NYLON --- BLOCKING BSA--- PRIMARY ANTIBODY INCUBATION--- WASHING-- SECONDARY ANTIBODY-- WASHING (5MIN*5). luciferase specific antibody used in 1;12000 dilution.

In vitro the promoter activity can be assayed using decanal( fatty acid aldehyde) will give a luminescent phenotype. The expression of lux Genes and promoter strength can be monitored in this way based on the level of bioluminescence produced.

Factors that regulate this operon in V fischeri are -

Population density( low levels at low cell density)