You have cloned the 4 bacterial species. Now you want to determine which one can degrade BA. You therefore, mix pure BA with these bacteria and wait for a day. Then you measure the decrease in BA levels in each bacterial culture. You find out that species #3 (Sp.3) reduces BA levels, and therefore, you decide that the gene which can degrade BA must be present in the genome of Sp.3. To find out which gene degrades BA, you make a microarray platform, with oligonucleotides corresponding to each gene of Sp.3. Then you perform the following experiment where you add the shown amounts of BA to Sp.3, and wait for the shown times, and you measure how much of BA is degraded. Results are shown in the below table. Experiment Nr. BA added Time waited BA remaining at the end of experiment 1 2 3 10ug 10ug 10µg th 6h 12h 10µg 24h 10ug 10ug 10ug 0.0001pg You then obtain mRNA from Sp.3 treated with BA for the times shown above and perform a microarray experiment. Some of the genes and the expression changes of these genes are shown below. Time 1h 6h 12h 24h Gene Nr. 1 20 2 2 2 2 50 75 100 50 3 250 1000 250 1000 4 35 37 39 1500 5 2 500 35 2 Which of these 5 genes would you pick to study further (clone and purify) as a candidate Base? Please explain your logic briefly. You picked one of the genes above and you now want to use it as a BAse. You, therefore, identify the ORF of the gene (1200bp), clone it into pQE30, and purify it using Ni-Cad, affinity chromatography. You then want to generate a monoclonal antibody against BAse. You therefore, inject the purified Base into mice, obtain their spleen cells and generate 5 hybridomas. To find out which of these hybridomas make correct antibodies to BAse, you perform the following experiment: you obtain bacterial lysates from Sp. 3, treated with BA, for 1h, 6h, 12h and 24h. You then run these lysates on an SDS-PAGE gel. You repeat this experiment for each of the 5 antibodies. & Last Name: a. Calculate the mw. of BAse (in kDs). Explain your logic very briefly. b. Please draw in the below empty image the bands you would expect to find for a monoclonal antibody that correctly recognizes BAse after Western blotting using the monoclonal antibody produced by the hybridoma.. Lanes: 1. Purified Base M.W.(KD) 2. Lysate (1h BA treatment) 3. Lysate (6h BA treatment) 200 4. Lysate (12h BA treatment) 100 5. Lysate (24h BA treatment) 50- 25 10- 5