Individual Final Project: Melanin-based light-recording bioink/biomaterial

Important links:

• Commited Listener Slide Deck here.
• Benchling (TO BE ADDED)
• Asimov Kernel (TO BE ADDED)

Aim 1: Build a first melanin-producing cell-free DNA module based on melA tyrosinase + Define validation parameters

The melA gene is coding sequence of tyrosinase that catalizes the conversion of tyrosine to dopaquinone. Dopaquinone is intermediate product of melanin biosynthesis pathway that polymerizes in an enzyme-independent reaction to form melanin.

The pathway from L-tyrosine to Melanin with the use of the melA tyrosinase.

1. Design MelA expression constructs (Benchling and Twist)

Some of the construct variables to be considered because it will affect melA expression:

• Promoter
• RBS
• Terminator
• Codon usage
• Tag placement
• Vector context

Note: a construct that works in cells may not translate well to cell-free expression.

Previous expression constructs:

• iGEM 2004 - using the P(lac)IQ promoter (BBa_I14032), a work designed by Vikram Vijayan, Allen Hsu, Lawrence Fomundam Group of the iGEM04 (2004-08-04 Part:BBa_I14032).

• iGEM 2009 - using B0040 RBS (composite part: BBa_K193602) containing pLacIQ(BBa_I14032), RBS(B0030) and melA(BBa_K193600) on low copy vector(BBa_I52001 derived), a work designed by Kazuaki Amikura.

• iGEM 2017 - Erin Kelly Group (2017-10-27) have developed a different expression construct specifically for use in E. coli BL21(DE3) which takes advantage of the T7 RNA Polymerase expression construct in the DE3 cassette to provide tighter epxression control and help to prevent leaky expression of the tyrosinase. Additionally, a double terminator B0015 was added to increase control over the system. In order to maximize production of the tyrosinase and limit unnecessary energy expenditure by the cell, a transcriptional terminator ensure energy is not wasted on transcribing an overly-long mRNA transcript. They transformed the melA_pJET plasmid into E. coli BL21(DE3) and attempted to overexpress the MelA tyrosinase (~54kDa) and produce the pigment melanin. Four colonies from the transformation were picked and used to produce pre-cultures, which were then used to incoulate test expression cultures. During the test expression, cultures were also supplied with CuSO4 and extra tyrosine. Cultures were induced with IPTG at OD600 ~1 and a 1OD sample was taken (T0). Another 1OD sample was taken after the cultures were left to grow overnight (TON). The cultures were allowed to grow another three days (supplemented with tyrosine and ampicillin) to see if pigment would form, but we were unable to detect any melanin. The 1OD samples were run on a 12% SDS-PAGE to check for melA overexpression (Figure 3). The MelA tyrosinase is ~54 kDa in size. A faint band of approximately 54 kDa appears in the TON lane of culture 3. This indicated that we were successful in expressing the MelA tyrosinase from the pJET plasmid. Before the Jamboree, we will attempt another overexpression of MelA from the pSB1C3 plasmid.

My take: T7 can maximize protein yield but also overwhelm folding capacity, causing inactive protein accumulation (increase the likehood of tyrosinases misfolds, aggregation, or fail to incorporate copper correctly). I’d replace it by a moderated construct and compare the results in reference to the BBa_K2481108 (control).

Note: MelA expression is not the same as melanin production. Melanin polymerization is messy, dopaquinone polymerizes through non-enzymatic downstream chemistry. This means color output depends not only on MelA, but also on oxygen, pH, time, redox state, and local chemistry.

2. Prepare reagents and workflow (Ginko & Open AI, HW 11C)

Melanin production in E. coli or in a cell-free system is influenced by several parameters that actuate at the level of melA expression and enzyme activity / posterior reactions:

• L-tyrosine concentration (substrate, limited solubility)

• CuSO4 concentration: since this tyrosinase is a type 3 copper-containing enzyme, Cu2+ is a cofactor of the enzyme. Too much copper can also stress cells or inhibit cell-free reactions.

• Magnesium

• Energy mix

• Molecular oxigen avaliability for tyrosinase reactions

• pH: tyrosinase activity and melanin polymerization are pH-dependent. If the reaction acidifies over time, enzyme activity or pigment formation may decrease.

Note: Optimizing for sfGFP may not optimize for MelA.

3. Validation

The 2017 iGEM result is a useful warning: they may have produced a faint ~54 kDa MelA band, but still detected no pigment. For this reason, I am proposing a staged validation workflow that moves from simple expression and pigment checks to more refined mechanistic analyses, depending on the results obtained in each previous round.

Results interpretation framework:

Here’s a diagram of my proposed validation workflow

4. Model a light-activated expression circuit that could later support gradual tonal change in a material system (Asimov Kernel).

Post-Course: Towards the melanin-based light-recording bio-ink

5. Refine Asimov Kernel’s output for controlling melanin expression till the system is fine-tuned with my aesthetical needs (most controlled and previsible) or, if not possible develop a experimental design for embedded sensing (in this case map both the quantitative and qualitative workflows available).**

Decision point: Push for maximum molecular control first, even if the material context is still abstract? OR Move earlier into material-scale experiments?

6. Material engineering: Test different models of integration of the melanin cell-free module towards a intended function/product. Tests for integrating into material though ELM engineer Komagataeibacter rhaeticus + bacterial celulose (BC) or a hybrid system with BC scaffold / other biomaterial that can be embed with cell-free modules with synthetic minimal cells / K. rhaeticus.

7. Rethink the workflow for measurable readouts for the cell-free system. RGB image analysis in a controlled lighting box or spectral (semi-quantitative) with melanin absorbance ranging from 475 nm to 500 nm;

8. Optimization rounds looking into every step of the proposed workflow, including economic tests for modeling parameters (such as ink dilution)

9. Benchmarking partnerships + reach out plan (heavly based on the previous item conclusions (BioFabricate, Cultivarium)