Open Ended Questions

1. Drew Endy’s
   1. How many people should have the option of learning to read and write DNA (i.e., practice biotechnology)
      1. I think that simple experiments concerning biotechnology should be taught universally, as a form to achieve a better understanding of how biological entities work in the general public.
   2. Pick a country outside the United States of interest to you. How does or will its bioeconomy differ culturally and practically from the US? How do or will these differences lead to differences in opportunities for specific biotechnology projects?
      1. I am from Argentina, and I think that the opportunities that there are great opportunities that biotechnology can offer my country and Latin America. As mostly agroexporters countries, we have tons of waste biomass that isn´t being used, the exploitment of this biomas via biotechnology, could help develope this countries economies.
   3. What is bio-scarer and does bio-scarer impact conversations about GMOs?
2. What is bio-scarer and does bio-scarer impact conversations about GMOs?

Learning module (Committed Listeners)

1. Patrick Boyle’s
   1. Assume that all of the molecular biology work you'd like to do could be automated, what sort of new biological questions would you ask, or what new types of products would you make?
      1. If this automatization was to happen, I think researchers should focus their efforts in areas that today demand too much timer or money. This could include, exploring complex biological systems, by changing multiple variables at a time and registering the outputs of this complex netxworks,personalized healthcare and agriculture by analizing each individual case and building on demand solutions, and finally exploration and analysis of enviromental genomes.
   2. If you could make metric tons of any protein, what would you make and what positive impact could you have?
      1. I thinkone promising option would be producing therapeutic proteins, such as monoclonal antibodies (mAbs). This mAbs can be use preventively or actively to deal with many diseases.

Bio Production Lab

1. Which genes when transferred into E. coli will induce the production of lycopene and beta-carotene, respectively?
   1. An additional enzyme is needed to produce beta-carotene.

1. Why do the plasmids that are transferred into the E. coli need to contain an antibiotic resistance gene?
   1. To ensure that the plasmid with the desired genes is continuously selected in the bacteria and doesn´t gets whashed up
2. What outcomes might we expect to see when we vary the media, presence of fructose, and temperature conditions of the overnight cultures?
   1. I would expect diferences in the final OD.
3. Generally describe what “OD600” measures and how it can be interpreted in this experiment.
   1. It measures the bacteria concentration in your culture, so that you can determine if the bacteria is on its exponential growth face or the stationary face for example.
4. What are other experimental setups where we may be able to use acetone to separate cellular matter from a compound we intend to measure?
   1. Acetone can be used to precipitate proteins from cell lysates or tissue homogenates. After cellular matter is disrupted and the compound of interest is extracted, acetone can be added to precipitate proteins, leaving the compound in the supernatant for further analysis.
5. Why do lycopene and beta-carotene have different absorption wavelengths?
   1. Because they are different molecules with double bonds, which absorbs wavelengths in the UV spectrum
6. Why might we want to engineer E. coli to produce lycopene and beta-carotene pigments when Erwinia herbicola naturally produces them?
   1. To achieve a higher production yield, by altering the genes involved, and using the vast knowledge available about E.coli growth.

Learning module (Committed Listeners)

1. Let's get in touch with our metabolic pathway

   1. What are the enzymes of the carotene pathway?

      1. The enzymes of this pathway are CrtE,CrtB,CrtL and CrtY.

      

   2. Within this pathway, which is the rate determining step (the step that takes the longest)? Which enzyme is responsible for this step?

      1. Generally, the first enzymes of a pathway, or the ones which demands a high ATP usage are the ones where there is a stronger control. In this case the CrtE seems to be the first enzyme of the methabolic pathway, so it´s probably the one regulated by energy availability and final product.
2. Notes for design of a DNA construct for bioproduction
   1. The first thing to do is to decide what organism you are going to use for this (E. coli or S. cerevisiae) for production. Which would you choose and why (emphases on production differences)?
      1. As the product of interest is a small molecule produced by a recombinant methabolic pathway y would choose E. coli as my chasis. My decision is based on the high growth rate of the bacteria, generating more biomass in less time and the ease to carry out genetic modifications.
   2. Now choose one of the enzymes and lets outline the parts of the construct for expression
      1. I choose CrtE
   3. For E. coli lets create a expression vector that works as a plasmid you choose E. coli let's create a expression vector that works as a plasmids
      1. Now, for making a functional construct there are a variety of biological parts needed for this, like ribosome binding sites, terminators, operators and promoters. The last ones are the most important in terms of enzyme or protein production. Let's elaborate further on this biopart.
         1. Promoter
            1. With the links below we are going to answer a few questions and think about the correct use of promoter: (https://blog.addgene.org/plasmids-101-the-promoter-region, https://www.addgene.org/mol-bio-reference/promoters/, https://blog.addgene.org/plasmids-101-repressible-promoters, https://blog.addgene.org/plasmids-101-inducible-promoters)

               1. What is the function of a promoter?

                  A promoter is a region of DNA located near the beginning of a gene, and its primary function is to initiate the process of transcription.The promoter serves as a binding site for RNA polymerase.

               2. What types of promoters do we have?

                  1. We can choose between consitutive promoters (constant rate of transcription), indicuble promoters (need to be turnedon by the addition of a molecule) or repressible promoters ( can be turned off by the addition of a molecule)

               3. If we wanted to turn off the transcription of a gene in response to a metabolite, what type of promoter would be most useful? What if we wanted this to increase in the presence of the metabolite?

                  1. In the first case we would need a repressible promoter, and in the second one we would need an inducible promoter.

               4. Now choose one of the genes of the metabolic pathway previously described (Carotene/lycopene )and choose one enzyme to make an expression construct. What promoter could you use for this? Why did you choose it?

                  1. I would use the promoter lac to regulate the gene CrtE. This allows me to let my bacteria reach the wanted DO and then turn on the methabolic pathway, so that sugars can be used to grow at first, but to synthesize the metabolites when bacteria are grown.
         2. Origin of replication of plasmid
            1. With the links below we are going to answer a few questions and think about the correct use of origin of rep: (https://blog.addgene.org/plasmid-101-origin-of-replication, https://blog.addgene.org/plasmids-101-plasmid-incompatibility, https://blog.addgene.org/plasmids-101-ebook-4th-edition)

               1. What is the origin of replication?

                  1. The origin of replication (ori) is a specific DNA sequence within a genome where DNA replication is initiated. In this case, it is the sequence that allows the plasmid we are using to replicate.

               2. What types of origin of replication do we have?

                  1. Control of replication is referred to as "relaxed" or "stringent" depending on whether the ori is positively regulated by RNA or proteins, respectively.

               3. (Extra) What are compatibility groups?

                  1. Compatibility groups indicate the plasmids that use the same machinery to replicate.As a rule, plasmids from the same group should not be co-transformed, so if you need two plasmids for an experiment, make sure they have "compatible" *ori'*s.

               4. Now for the previously chosen promoter and gene what will be the best origin or replication?

               5. I would usea a relaxed control plasmid with not a high number of copies (I don´t want energy to be wasted in creating DNA molecules) such as pColE1

         3. (Mandatory for Global listeners, Optional MIT/Harvard) Elaborate further on other bioparts like RBS, terminators, operators you would use for a correct design and further bioproduction?
            1. I would need to introduce a RBS for prokaryotic translation, so the Shine-Dalgarno sequence need to be added to my gene. A prokaryotic terminator is also needed as the original genes come from plants; i would choose a terminator rho-independant to not increase the complexity of my constructs.
         4. (Hot! Extra points) What are aptamers and riboswitches and how can they be used for metabolic tuning or engineering in prokaryotes?

            1. Aptamers and riboswitches are two types of RNA molecules that can be utilized for metabolic tuning or engineering in prokaryotes to regulate gene expression or metabolic pathways in response to specific ligands or conditions.

               1. Aptamers are short, single-stranded RNA molecules that can fold into specific three-dimensional structures, allowing them to bind tightly and selectively to target molecules like RNA antibodies. They can be introduced into the regulatory region of the gene, to allow the regulation of RNA concentration by desired metabolites.

               

               1. Riboswitches are structural elements found in the untranslated regions (UTRs) of certain mRNAs that can directly bind small molecule ligands and regulate gene expression in response to ligand binding.Upon ligand binding, riboswitches can adopt different conformations that either promote or inhibit gene expression, depending on the specific riboswitch and the context of the gene it regulates.
      2. (Extra points) Now what approach can be used to join all these parts together? Make a quick analysis of their sequence in search of possibilities (search for restriction sites, etc)
      3. (Extra Hot!!! Extra Points) Try to elaborate further on a biosynthetic pathway you would want to engineer in E. coli for production of a metabolite or product. What use could this bio-product have? Imagine dream applications!!!
   4. (Extra points) For S. cerevisiae create an integration cassette for homologous recombination.
      1. First let's check some concepts of yeast engineering and homologous recombination this in this notes
      2. As well as for prokaryotes, eukaryotic DNA designs need bioparts used for construction of a function design and further expresion. Now search for a biosynthetic pathway if interested and describe one of the genes of the pathway.
      3. Now, remember that for making a functional construct there are a variety of biological parts needed for this, like ribosome binding sites or Kozak sequences, terminators, and promoters. List the ones you could use for DNA design.
      4. In yeast engineering we use DNA construction designs for making genome integration. What chromosome site could you use for integration of these and why?
      5. (Hot! Extra points) Following the next chart of how a DNA integration cassette should be designed and with the previously chosen parts elaborate the DNA sequence you could use to synthesize with Twist.