Homework
Weekly homework submissions:
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?
Week 2 HW: DNA read, write and edit
Homework Part 1: Benchling and in-silico gel art This is the virtual digestion of the Lambda genome with this restriction enzymes: EcoRI, HindIII, BamHI, KpnI, EcoRV, SacI and SalI.
Homework I create the following code by drawing at the opentrons art platform, coping the coordenates and asking chatgpt to generate the code below the code provided by the original opentrons HTGAA google colabs. Colabs link mclover3_points = [(-27, 24),(-24, 24),(-21, 24),(-18, 24),(-30, 21),(-27, 21),(-18, 21),(-15, 21),(-3, 21),(0, 21),(-33, 18),(-30, 18),(-15, 18),(-12, 18),(-3, 18),(0, 18),(-33, 15),(-30, 15),(-24, 15),(-21, 15),(-18, 15),(-12, 15),(-3, 15),(0, 15),(-33, 12),(-27, 12),(-24, 12),(-21, 12),(-18, 12),(-15, 12),(-3, 12),(0, 12),(-33, 9),(-27, 9),(-24, 9),(-12, 9),(-3, 9),(0, 9),(-30, 6),(-9, 6),(-6, 6),(-3, 6),(0, 6),(-33, 3),(-3, 3),(0, 3),(-6, 0),(-3, 0),(0, 0),(15, 0),(18, 0),(21, 0),(24, 0),(27, 0),(-3, -3),(0, -3),(12, -3),(15, -3),(18, -3),(24, -3),(27, -3),(30, -3),(-3, -6),(0, -6),(9, -6),(12, -6),(21, -6),(33, -6),(-3, -9),(0, -9),(9, -9),(15, -9),(18, -9),(21, -9),(24, -9),(27, -9),(30, -9),(-3, -12),(0, -12),(12, -12),(15, -12),(18, -12),(21, -12),(24, -12),(27, -12),(30, -12),(-3, -15),(0, -15),(3, -15),(6, -15),(12, -15),(15, -15),(18, -15),(21, -15),(24, -15),(27, -15),(-3, -18),(0, -18),(-3, -21),(0, -21),(-3, -24),(0, -24),(-3, -27),(0, -27),(-3, -30),(0, -30),(-3, -33),(0, -33),(-3, -36),(0, -36),(-3, -39),(0, -39)] sfgfp_points = [(-24, 21),(-21, 21),(-27, 18),(-24, 18),(-21, 18),(-18, 18),(-27, 15),(-15, 15),(-30, 12),(-12, 12),(-30, 9),(-9, 9),(-33, 6),(-6, 3), (21, -3),(15, -6),(18, -6),(24, -6),(27, -6),(30, -6),(12, -9),(33, -9),(6, -12),(9, -12),(3, -18)] electra2_points = [(-3, 24),(0, 24)] def draw_points(color, points): DISPENSE_VOL = 0.75 pipette_20ul.pick_up_tip() for x, y in points: pipette_20ul.aspirate(DISPENSE_VOL, location_of_color(color)) adjusted_location = center_location.move(types.Point(x=x, y=y)) dispense_and_detach(pipette_20ul, DISPENSE_VOL, adjusted_location) pipette_20ul.drop_tip() draw_points('Green', mclover3_points) draw_points('Red', sfgfp_points) draw_points('Orange', electra2_points) The simulation provide the following artwork.
Week 4 HW: Protein desing part I
Homework Part A. Conceptual Questions Answer any NINE of the following questions from Shuguang Zhang: Why do humans eat beef but do not become a cow, eat fish but do not become fish? Because eating beef doesn´t make us incorporate the genetic information that make a cow, and even if we could, we don´t have machinery to process that information and make us a cow.
Week 5 HW: Protein design part II
Homework Part A: SOD1 Binder Peptide Design (From Pranam) Part 1: Generate Binders with PepMLM 1- Begin by retrieving the human SOD1 sequence from UniProt (P00441) and introducing the A4V mutation.
Week 6 HW: Genetic Circuits Part I: Assembly Technologies
Assignment: DNA Assembly What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose? The high Phusion High-fidelity polymerase, MgCl2, dNTPs, etc. The polymerase enable low error in the polymerization reaction. What are some factors that determine primer annealing temperature during PCR? how much Hidrogen bombs are present (type of bases and how much).
Week 7 HW: Genetic Circuits Part II: Neuromorphic Circuits
Assignment Part 1: Intracellular Artificial Neural Networks (IANNs) What advantages do IANNs have over traditional genetic circuits, whose input/output behaviors are Boolean functions? It enable non-linear threshold computation over the concentration of the molecules. Describe a useful application for an IANN; include a detailed description of input/output behavior, as well as any limitations an IANN might face to achieve your goal. An useful application could be the development of responses upon specific thresholds of morphogens, for long-space tissue patterning (and cell-fate determination).
General homework questions Explain the main advantages of cell-free protein synthesis over traditional in vivo methods, specifically in terms of flexibility and control over experimental variables. Name at least two cases where cell-free expression is more beneficial than cell production. Cell-free protein synthesis enable rapid protein production without the need to wait for traditional transformation and growth. This fast prototyping enable fast test of multiple genes at the same time, genetic parts, variants, and other kind of libraries. Also, other variables, such as temperature, concentration of analytes, non-canonical AAs, etc, could be tested easily in CFE. One case could be when characterizing multiple parts in an organism, where making it in CFES would be much more time efficient. Other case could be when making proteins with non-canonical AAs, making it more easy in the CFES.
Week 10 HW: Advanced Imaging & Measurement Technology
Waters Part I — Molecular Weight Based on the predicted amino acid sequence of eGFP and any known modifications, what is the calculated molecular weight? The calculated molecular weight of eGFP with the LE linker and His-tag is approximately 27.1 kDa. Determine z for each adjacent pair of peaks using: Using adjacent peaks around 933.7 and 1000.4 m/z, the charge state is approximately +15.
Week 11 HW: Bioproduction & Cloud Labs
Part A: The 1,536 Pixel Artwork Canvas | Collective Artwork I couldn´t contribuite to the pixel artwork sadly :( Next year i become a TA without excuses. Part B: Cell-Free Protein Synthesis | Cell-Free Reagents Referencing the cell-free protein synthesis reaction composition (the middle box outlined in yellow on the image above, also listed below), provide a 1-2 sentence description of what each component’s role is in the cell-free reaction. E. coli Lysate: Provides the cellular machinery for transcription and translation, including ribosomes, tRNAs, enzymes, and metabolic proteins.
Lab questions Which genes when transferred into E. coli will induce the production of lycopene and beta-carotene, respectively? The production of lycopene in E. coli is induced by the genes crtE, crtI, and crtB from Erwinia herbicola, which are carried in the pAC-LYC plasmid. Beta-carotene production requires the same pathway plus the additional crtY gene, present in the pAC-BETA plasmid.