If all molecular biology work could be automated, I would like to focus on biological questions regarding the evolution of bacteria. I believe that there is much more to discover regarding evolution patterns within bacterial cell cultures at different conditions. The limitation is that these type of experiments require intensive handling by the researchers, in order to maintain the culture conditions fresh and optimal. This handling can result in contamination or could be messed with by human error. If a machine can be programmed to be able to maintain cellular cultures for various generations, limiting the researchers’ interactions with the culture and human error, it is more likely for it to succeed. Specifically, I think that experiments regarding bacterial degradation of contaminants, being those in water or soil, can provide many benefits in the field of biorremediation.
If I could make metric tons of a protein I would chose one that is used in the medical field, as a treatment for a disease. One example of this is the production of insulin; currently it is being produced by microorganisms in order to try and meet the demand for it. There are other proteins, like antibodies for vaccines and different treatments that need to be produced to prevent or treat diseases. I believe that this application would have the most impact within society.
For the production of lycopene in E. coli, the pAC-LYC plasmid will be inserted. This plasmid has the genes to produce the enzymes geranylgeranyl diphosphate synthase (CrtE), phytoene synthase (CrtB) and lycopene beta-cyclase (CrtL), which are responsible for the biosynthetic pathway that produces lycopene from fransesyl diphosphate. In order to produce beta-carotene in E. coli, the plasmids pAC-LYC and pAC-BETA will be inserted. Therefore, this strain will have the genes for the lycopene pathway (CrtE, CrtB, CrtL) and an additional gene CrtY for the production of the enzyme beta-carotene hydroxylase.
The plasmids pAC-LYC and pAC-BETA contain the gene for the antibiotic chloramphenicol resistance in order to positively select the cells that have recived both plasmids with the biosynthesis pathway for lycopene and beta-carotene.
The two media tested in this lab is LB and 2YT. Both are enrichment media, which means that they promote cell growth. However, 2YT is richer than LB and has been designed specifically for the growth of E. coli bacteria. The medium 2YT has typtone and yeast extract that provide a wider range of nutrients that a cell can use for food, activating different pathways in order to degrade the compounds, maintaining the cell active. Therefore, it is expected that the samples cultured in 2YT media grow more than those in LB.
Fructose is a simple sugar that can also be used by bacteria as a source of carbon to create energy. Therefore, the samples cultivated with fructose should present a greater growth than those without this sugar.
The two temperatures of incubation of the different samples are 30°C and 37°C. E. coli is a mesophyl bacteria, which means that its optimal temperature for growth is 37°C. At this temperature, E. coli multiplies every 20 minutes and has a very active and fast metabolism. When transforming this bacteria for the production of recombinant proteins, some are not properly expressed when E. coli is working at its optimal rate. Since the metabolism is very active, some errors in translation may occure or the protein does not fold properly, resulting in a non-functional product. By incubating it at a lower temperature, the growth slows down and recombinant proteins have more time to fold into the correct shape.
OD600 is a measurement that evaluates the optical density of a sample at 600 nm of a light wave. This is measured by a spectrophotometer, which shoots a 600 nm beam of light through a cuvette with the sample and it records the amount of light that is absorbed by the matter within the sample. More light absorbtion is caused by a bigger concentration of matter in the sample, which in this case is the amount of cells that grew. Therefore, the amount of light absorbtion is directly related to the concentration of cells in the medium, which can be used to form a line used to calculate the exact concentration of cells. Specifically, the OD600 measurement uses a graph that has been constructed for E. coli. If the experiment would require to measure the concentration of cells using optical density for other types of bacteria, the wavelength used would be different and specific to the bacteria measured.