Week 1 HW: Principles and Practices

Project Description

I want to develop a synthetic assymetrical-cell-division system to enable synthetic cell differentiation; as a toolkit for engineer multicellular organization, development, pattern formation and others. To futher detail, see the page of the final project

The project have two major subprojects:

• A mitotic counter: a circuit capable of counting as states how many cell division the linage have sense since the system has been activated (firts division, second, third, and so on). The system uses the natural fluctuations of the cell’s cycle regulators to induce or activate distinct proteins. When the system is activated, a TF would be activated by an cell-cycle-dependent kinase (at the G1 phase), that would induce the expression of a recombinase, that would inverse the sequence of its own promoter, inducing the expression of an inactivated, second, TF. This TF2 would be activated via a second cell-cycle-dependent kinase, one that would be activated in the final of the cell cycle and not be expressed at the same time with the firts kinase. The phosphorylation upon TF2 would, for example, link two homodymers together, in an activated form (an option, but not necessarily how it would work). When the kinase 2 activates TF2, gene expression would not be available because of chromatin condesation upon mitosis. After the cell divides, at G1, TF2 could induce the activation of other genes, that, in consenquence and, using the architecture, enables the activation of other genes at the second cell division, and so on, making possible to count cell division in the cell lineage. The system also needs other things such as a degradation system, repression for past states, etc.
• Assymetrical component: ¿How can we engineer an assymetrical distribution of molecules to induce the assymetrical cell division? It should be transferable to other organisms. The proposed one is to used an synthetic phased segregated condensate that is capable of create one individual and stable condensate upon the cell. It should carry an mRNA that would express a TF. It should be formated upon the activation of the scaffolds and dissegregated upon the cell division, enabling the translation of the mRNA selected. The mRNA should be sequestred in the condensate, where its translation-initiation site would be blocked, and it would be protected of degradations by directed nucleases over that mRNA. All of this via linking the sites of union of the scaffolds with this important sites of the mRNA (nuclease recognition site, translation initiation site, rybozome binding site, etc). The scaffolds should be inactivated upon cell division via phosphorylation at the binding sites, liberating the mRNA. But how we can make a single condensate exist? The idea is to use the pyrenoid (a rubisco aggregate condensate that makes CO2 fixation more efficient) as an example for this, where, in a dynamics between phosphatases and kinases, the algae maintains the pyrenoid as a single condensate or multiple when needed.

¿So how all of this is going to work in order to make the assymetrical cell division system function?

When both systems are activated, the mitotic counter and the phase-condensate system, the single condensate is going to be segregated to any of the son’s cell, and, after the division have been made, the TF2 of the mitotic counter could express a kinase for the dissegregation of the condensate, enabling the translation of the mRNA. Also the system could make possible the use of logic gates, whether both components are present or no, x gene is activated (in the other cell that would have only the TF2 expressed, AND, NOT or OR gates based on this could enable the differentiation in this cell also, taking another fate that the father cells haved. Or even more complex differentiation dynamics). Anyways, the now different cell could express an auto-induced transcription regulator, that, as follows, would induce the differentiation of its own self, and his son’s cells (between other feedbacks). Also the mRNA TF should repress the component of mRNA degradation.

¿And now what?

Those systems are toolkits for the multicellular engineering dicipline. To directly create organisms, we would need to have an autonomous cell differentiation system, a system that by its own is able to activate itself. The mitotic counter, also, for example could work as a signaling event upon certains cell divisions in the designated cell lineage, to activate cell-to-cell communications upon that moment, and only in that moment for example. Possibilities are endless and only the imagination is the limit.

Governance Context

Autonomous multicellular systems capable of growth, differentiation, and spatial organization introduce additional biosafety and biosecurity considerations compared to single-cell engineering. Risks include unintended environmental persistence, uncontrolled proliferation, ecological interaction, and potential dual-use of self-assembling biological structures. Governance must therefore balance intrinsic biological containment, institutional oversight, and research feasibility.

Options considered

Option 1 — Mandatory Genetic Containment and Fail-Safe Design
Intrinsic safeguards such as auxotrophy, kill-switches, replication limits, and environmental sensitivity.

Option 2 — Project Licensing, Design Review, and Traceability
Registration, documentation, biosafety review, strain tracking, training, and incident reporting.

Option 3 — Voluntary Standards and Community Best Practices
Non-binding guidelines and self-governance without formal enforcement.

Scoring scale: 1 (low contribution) to 5 (strong contribution).

Homework pre HW2:

Answers to Professor Jacobson

DNA replication is carried out by DNA polymerases, which exhibit a raw error rate of approximately 10⁻⁵ errors per base incorporated. However, proofreading activity and mismatch-repair pathways dramatically improve fidelity, reducing the effective error rate to roughly 10⁻⁹–10⁻¹⁰ per base.

Given that the human genome contains about 3 × 10⁹ base pairs, an uncorrected replication process would introduce tens of thousands of mutations per cell division. In practice, only a few or fewer mutations accumulate per division. This discrepancy is resolved through polymerase exonuclease proofreading and post-replication DNA repair systems that detect and correct mismatches before they become permanent mutations.

Because the genetic code is degenerate, most amino acids are encoded by multiple synonymous codons (on average roughly three per amino acid). Consequently, a typical human protein of several hundred amino acids could theoretically be encoded by an astronomically large number of different DNA sequences. In practice, however, only a small subset of these sequences functions efficiently. Constraints include codon bias and tRNA availability, unfavorable mRNA secondary structures, extreme GC content, unintended splice or regulatory signals, reduced translation efficiency, and technical limitations in DNA synthesis or cloning. These factors strongly restrict the number of sequences that yield robust protein expression.

Answers to Professor LeProust

The most widely used method for oligonucleotide synthesis is solid-phase phosphoramidite chemical synthesis. In this approach, DNA is assembled one nucleotide at a time on a solid support through automated cycles of nucleotide coupling, washing, and deprotection. This chemistry has remained the industry standard for decades because it is fast, scalable, and reliable for producing short DNA fragments.

Oligos longer than approximately 150–200 nucleotides are difficult to synthesize efficiently because each coupling step has slightly less than perfect efficiency. A small fraction of strands fails to extend at every cycle, and these losses accumulate exponentially as the number of cycles increases. As a result, the final mixture contains many truncated or error-containing products, while the yield of full-length DNA becomes very low. Additional chemical side reactions and base damage further degrade quality.

For this reason, a 2000 base-pair gene cannot be synthesized directly. The cumulative inefficiency across thousands of addition steps makes the probability of obtaining a correct full-length product essentially zero. Instead, long genes are constructed by synthesizing many shorter oligonucleotides and assembling them enzymatically using methods such as PCR assembly or Gibson assembly.

Answer to Professor Church

In most animals, including humans, nine amino acids cannot be synthesized de novo and must be obtained from the diet: histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. Some sources include arginine as conditionally essential, particularly in growing organisms, yielding a list of ten.

Because lysine is already universally essential, engineering a synthetic “lysine contingency” does not create a fundamentally new biological vulnerability. Instead, it exploits an existing metabolic dependency. Organisms would already require external lysine, so forcing supplementation simply makes this requirement explicit rather than introducing a novel Achilles’ heel. Consequently, such a contingency is predictable and limited as a containment strategy rather than uniquely robust.

Week 2 HW: DNA read, write and edit

Homework

Week 1 Lab: Pipetting

Individual Final Project

Group Final Project