5. Generally we clone from cDNA libraries, not from a host genome--it's just simpler. However, pseudomonas is really not a big deal containment-wise. Most strains require no special handling aside from what you are already doing. I once took a shower in about 50 gallons of pseudomonas areuginosa culture when my fermenter spewed all over me. My lab worked with bacteria that degraded phthalates and other complex organic molecules. P. areuginosa is an opportunistic bacteria, but various strains of pseudomonas are on us and around us all the time. Unless you're looking at a known pathogenic strain, you don't need higher biosafety precautions.
6. When you are talking about a cloned gene, expression is always controlled by the plasmid. The promoter on the plasmid controls gene expression, and it is almost always constitutive--always on. It will not "turn on" or "turn off" in response to the presence of melamine in the culture. In some biological systems, the substrate of a reaction will act as a promoter for a gene for a metabolic enzyme--but only sometimes, and only in the native organism. Once you clone the gene into another organism, there's not going to be a connection between substrate and gene expression.
7. Be careful when you heat agarose. It has a tendency to superheat and boil over. Add it to your buffer in a generously sized erlenmeyer flask, cover the flask loosely with saran, and microwave it for about 30 seconds at a time, then swirl and check to see if it's dissolved, using a heavy-duty heat-and-water proof glove (not an oven mitt).
8. What you are doing is basically 1970's technology. That is when molecular biology was just getting started and a lot of biochemists were kludging together equipment and supplies much as you are. If you check out methods manuals and seminal papers from the 1970's, you'll find pretty straightforward methods for building equipment and putting together apparatus. Even back in the early 90's, I can remember pouring my agarose gels into frames made out of tape. Acrylamide is extremely toxic. Handling it is not so bad, if you're smart enough not to eat it. But you will be generating toxic waste and you can't flush that down your drain. You will have to find an acceptable way to dispose of your toxic waste. Pouring PAGE gels is very tricky.
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6. When you are talking about a cloned gene, expression is always controlled by the plasmid. The promoter on the plasmid controls gene expression, and it is almost always constitutive--always on. It will not "turn on" or "turn off" in response to the presence of melamine in the culture. In some biological systems, the substrate of a reaction will act as a promoter for a gene for a metabolic enzyme--but only sometimes, and only in the native organism. Once you clone the gene into another organism, there's not going to be a connection between substrate and gene expression.
7. Be careful when you heat agarose. It has a tendency to superheat and boil over. Add it to your buffer in a generously sized erlenmeyer flask, cover the flask loosely with saran, and microwave it for about 30 seconds at a time, then swirl and check to see if it's dissolved, using a heavy-duty heat-and-water proof glove (not an oven mitt).
8. What you are doing is basically 1970's technology. That is when molecular biology was just getting started and a lot of biochemists were kludging together equipment and supplies much as you are. If you check out methods manuals and seminal papers from the 1970's, you'll find pretty straightforward methods for building equipment and putting together apparatus. Even back in the early 90's, I can remember pouring my agarose gels into frames made out of tape. Acrylamide is extremely toxic. Handling it is not so bad, if you're smart enough not to eat it. But you will be generating toxic waste and you can't flush that down your drain. You will have to find an acceptable way to dispose of your toxic waste. Pouring PAGE gels is very tricky.