genome
engineering
CLASS ASSIGNMENT
THEORETICAL HOMEWORK
Questions
1. Craig Venter has stated he will establish an iGEM style competition for the most innovative use of the JCVI minimal bacterial cell, JCVI-syn3.0. The JCVI built the organism to be used as a platform to investigate the first principles of cellular life. Rather than use the cell for a basic research question, imagine that you were head of R&D at a biotech and were trying to determine if a new biochemical pathway you had developed to fix carbon from CO2 had any industrial potential. You know the method works in cell free enzymatic reactions, but that is about all you are sure of other than the enzymes and pathway. Do not get stymied by technical details involving how you would genome engineer JCVI-syn3.0. Explain the concept and experimental rationale.
For scaling up the biochemical pathway in an industrial application, we can follow the traditional Design, Build, Test cycle.
The jump from basic science to applications is challenging, because there are many parameters that have to be taken into consideration. The first question I would ask myself is who would benefit from this, i.e. who would want to pay for it. Then, the question becomes where to capture the CO2 to serve this customer best. Is the goal to perform direct-air capture or strategically at positions with a high concentrations of CO2, such as the exhausts of industrial factories? Or perhaps as a new carbon capturing system for cars? It is important to define who the owner of the problem is and what her or his interests and requirements are, then you can start the engineering process with a clear end goal in mind. Market research that includes an exhaustive evaluation of other carbon capturing techniques and organizations (such as Climeworks, CO2 Solutions, Carbon Engineering, PHYCO2) could help too. A helpful resource in shaping the business plan is the circular design guide, launched by the design firm IDEO. This contains tools and templates for organizations that aspire circular economy (CE) propositions and helps in the shift from a focus on product to service.
Following the business strategy, the technical aspects of scaling up to an industrial application can be determined. It is necessary to obtain knowledge about the conditions under which JCVI-syn3.0 is capable of fixing carbon. This can be done by studying scientific research papers and by experiments. The biochemical pathway to build CO2 converting pathways works in laboratory settings, but has to be tested under under other circumstances. Small scale bioreactors can be used for fast and high-throughput testing of independent environmental variables. The main challenge I foresee is related to how the bacterial minimal cell system reacts to fluctuations. During the day and night, summer and winter, carbon levels fluctuate. How does the cell respond to those changing supplies of carbon dioxide, and shifts in temperature, humidity, etc.? What are the conditions for maintaining life and quality? In addition to experiments, in silico computer simulations can provide more insight in the parameters that influence the I/O of bioreactors. The last questions to ask are; what happens with the captured CO2? How is it extracted, stored and transported, in solid or liquid form? Will it be injected in designated underground storage locations or used as an input source for micro-algae production and greenhouses?
2. In previous classes, George Church has talked about work done in his lab to do grand scale genome engineering of E. coli to alter its genetic code. While there are similarities with how his lab engineers E. coli with how the JCVI engineers mycoplasmas, what is the fundamental difference or differences?
Similarities are that both labs follow the design-build-test cycle.
However, the Church lab engineers the genome of E.coli top-down,
by sequential deletions of the genome. After each deletion, viability growth rate and other phenotypes are determined. In contrast, the JCVI used a bottom-up approach to engineer mycoplasmas from a reduced genome. First, a hypothetical minimal genome (HMG) was determined based on a combination of existing transposon mutagenesis, deletion data and literature research. Then the design, chemical synthesis, and assembly of bacteria with reduced genomes follows.
The Church lab uses CRISPR and Multiplex Automated Genome Engineering (MAGE) for genome engineering. MAGE is also called accelerated directed evolution. The technology is capable of taking a population of cells and adding, deleting, and replacing DNA sequences at very specific target locations within the cellular genome. [1].
3. In the Science paper “Design and synthesis of a minimal bacterial genome,” the JCVI showed the figure below, which is the first step in an effort to rationally reorganize the minimal cell genome. As noted in the paper, when we reorganized the genome, which included separating genes in operons, as needed we placed genes behind transcriptional promoters that controlled expression of genes deleted to build minimized segment 2. We built a genome that had a reorganized segment 2, but with the other 7 segments in with their original gene order. As noted in the paper, that cell grew normally. In later experiments, the JCVI used the same strategy to design and build reorganized versions of the other 7 minimized segments and tested them as genomes that were 1/8th reorganized and 7/8ths not reorganized. None of those 7 genomes resulted in viable cells. What is your hypothesis as to why this happened?
According to the paper, the following types of genes can be deleted:
1. One out of two identical genes that perform the same function
2. One out of two identical genes that both perform an essential function, but we don't know what that function is
While the benefits of reducing an organism's genome for large scale production of biofuels are obvious, my main reservation is as follows. When you remove 'junk' DNA from any organism, what functions are removed? The scientific name is satellite DNA, representing the part of the genome that does not encode proteins. Scientists still do not comprehend its function. But does that mean we can get rid of it? The University of Michigan had the same question. They used a common model organism, the fruit fly, and mouse cells to study the function of these long, repetitive satellite sequences. It turns out that satellite DNA plays a crucial role in holding the genome together. Without it, they observed chromosomal instability in the form of micro-nuclei formation, DNA damage and cell death. This might be an explanation for the non-viability of JCVI's seven genomes.
Reorganization of gene order in JCVI-syn3.0 segment 2. Genes involved in the same process were grouped together in the design for “modularized” segment 2. At the far left, the gene order of syn1.0 segment 2 is indicated. Genes deleted in syn3.0 are indicated by faint gray lines. Retained genes are indicated by colored lines matching the functional categories to which they belong, which are shown on the right. Each line connects the position of the corresponding gene in syn1.0 with its position in the modularized segment 2. Black lines represent intergenic sequences containing promoters or transcriptional terminators.
4. The JCVI recently announced that it was now minimizing and reorganizing the genome of the fastest growing eukaryote, a yeast called Kluyveromyces marxianus. The goal will be to make an alternative to Saccharomyces cerevisiae that grows faster, and can be grown at higher temperature, and that would be a better platform for the kinds of biotechnology people currently use yeast for. Based on the “genome engineering lecture” in HTGAA and the Science paper “Design and synthesis of a minimal bacterial genome” (assigned as class reading), how would you go about minimizing and rationally reorganizing the K. marxianus? What would be different about your approach relative to how the JCVI minimized Mycoplasma mycoides to produce JCVI-syn3.0?
To answer this question, I would find out what the differences are between the yeast Kluyveromyces marxianus and bacteria Mycoplasma mycoides. The yeast is eukaryotic and has a more complex genome than the E.coli bacteria. Based on the paper presented in question 3, I would do research to what the functions of the non-coding regions in its DNA are.