GENOME ENGINEERING
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.
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?
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.
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.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. After reading the 2016 JCVI paper “Design and synthesis of a minimal bacterial genome” and Harold Morowitz’s 1984 essay “The completeness of molecular biology” list a few of your thoughts about how Morowitz’s thinking affects modern thinking about minimal cells and life. What did you like or not like about the Morowitz essay?
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?
We can introducing a metabolic pathway from plants into the JCVI minimal bacterial cell to do that. This pathway is part of photosynthesis and able to fix carbon dioxide (CO2). The carbon is then converted by the bacteria into carbon-containing biofuels or other economically important products.
A fundamental difference is the approach for genetic modification. While George Church´s team are trying to do grand scale genome engineering of E. coli to alter its genetic code from an existing genome. The JCVI group´s approach creates genome sequence from zero in order decrease the amount of genes in the cell.
I like how Morowitz influenced the thinking of many scientists suggesting that the logic of life can be written in 600 steps. Completely understanding the operations of a prokaryotic cell is a visualizable concept, one that is within the range of the possible. However we can’t underestimate the complex of Biological systems and the limitations of that.
Biological systems are extremely complex and have emergent properties that cannot be explained, or even predicted, by studying their individual parts.
There was one more intriguing element to Morowitz’s plan:
At 600 steps, a computer model is feasible, and every experiment that can be carried out in the laboratory can also be carried out on the computer. The extent to which these match measures the completeness of the paradigm of molecular biology.
Looking back on these proposals from the modern era of industrial-scale genomics and proteomics, there’s no doubt that Morowitz was right about the feasibility of collecting sequence data. On the other hand, the challenges of writing down “the logic of life” in 600 steps and “completely understanding” a living cell still look fairly daunting. And what about the computer program that would simulate a living cell well enough to match experiments carried out on real organisms?
The minimal cell concept appears simple at first glance but becomes more complex upon close inspection. In addition to essential and nonessential genes, there are many quasi-essential genes, which are not absolutely critical for viability but are nevertheless required for robust growth. The reason why none of those 7 genomes resulted in viable cells is that there was a need of quasi-essential genes present in the segment 2.