Balloons flown in Earth’s stratosphere are offered limited UV protection due to thinning of the atmosphere with increasing altitude. Chief Scientist of World View Enterprises Alan Stern stated in a meeting with our team that the materials used in his near-space exploration vessels cannot be reused due to the radiation damage they sustain during flight. The Stanford Space Initiative’s Balloon team expressed to members of our team that radiation exposure was the most significant factor in limiting the lifespans of their balloons. The unmet need for reusability of balloon materials flown in high altitude conditions generates a lack of sustainability that our team aimed to tackle through UV resistance.

On Earth, the main source of concern for radiation damage is ultraviolet radiation (UVR). These wavelengths are between 290 and 400 nm when they hit the Earth’s surface, and do more damage the shorter they get. UV wavelengths between 320 and 400 nm are called UV-A, and are the culprits behind sunburns and cataract formation. UV-A radiation creates indirect DNA damage through the generation of reactive oxygen species (ROS), forming strand breaks in DNA and DNA-protein crosslinks. [1] Shorter wavelengths from 290 to 320 nm are called UV-B wavelengths. UV-B is absorbed directly into DNA and causes damage to DNA at the molecular level. [2] Excessive UV-B exposure can induce significant DNA mutation and cell death, and our bioballoon will be exposed to dangerous amounts of radiation due to its high elevation during flight. Ideally, our balloon coating would have included an interweaving of zinc oxide, a white pigment that excels at blocking and reflecting UV-A and UV-B rays. [3] Zinc oxide is used in many commercial sunscreens, as a solution with as low as 25% zinc oxide content results in protection of at least SPF 20. [4] However, we wanted to produce our UV protector entirely biologically rather than attempting to incorporate an inorganic compound into our membrane. As such, we decided to focus on creating a method of UV protection for our balloon that utilizes a melanin incorperated via a binding agent directly into our membranes.

Genomic AroG and AroF are inhibited in vivo by the presence of phenylalanine and tyrosine respectively, both of which are synthesized through the shikimate pathway. [9] In AroG, two single codon point mutations (Pro150Leu and Leu175Asp) can be altered to generate feedback resistant forms of this isozyme. KU Leuven’s 2013 iGEM team biobricked the natural AroG enzyme and identified the same two mutation sites (found here BBa_K1060000). Accordingly, our team ordered the part from iGEM headquarters, and designed primers to perform site directed mutagenesis at these two locations. Before biobricking these parts, we inserted our two AroGfbr mutants into a pSB1C3 backbone derived from our composite rAIP plasmid ( BBa_K2027000), which included a constitutive promoter, strong RBS, and double terminator that flanked our genes. A similar point mutation to wild-type AroF (Asn8Lys) can instill tyrosine feedback resistance in the enzyme. [10] Having never previously been biobricked, we ordered our AroFfbr gene as two gBlocks from IDT and assembled the construct using Gibson Assembly Protocol into the same composite backbone as our AroGfbr genes.

To further stimulate the shuttling of carbon into the shikimate pathway, we aimed to decrease the metabolic consumption of PEP and E4P, the substrates of DAHP synthase. PEP and E4P are consumed by the glucose phosphotransferase transport system (PTS). Inactivation of this system has been shown to increase the yield of these compounds in E. coli grown in a glucose medium. [11] Instead of spending hours in the lab performing genomic deletions in our host cells, we contacted Dr. Guillermo Gosset of La Universidad Nacional Autónoma de México in request of a live sample of his PTS- tyrR- glucose+ strain VH33tyrR. [10] This strain, derived from W3110 E. coli contained genomic knockouts of PTS and the tyrR negative transcriptional regulatory protein, decreasing the cellular consumption of PEP and E4P and increasing the biosynthesis of and transport of tyrosine. Dr. Gosset readily agreed, sending us samples of his VH33tyrR strain lacking an AroGfbr containing plasmid pRW300, which he could not distribute due to patent regulations. He sent us a secondary sample of W3110 E. coli containing his pMmelAtyrCpheACM plasmid, which included genes for the tyrC protein and mutMelA tyrosinase necessary for melanin production. We extracted the plasmid from the secondary cell sample with a Miniprep kit, but ran into vast difficulties transfecting his VH33tyrR cells with his plasmid and our engineered AroGfbr and AroFfbr plasmids. After numerous attempts at chemical transformations and electroporation (see our lab notebook), we settled on transfecting T7 Express E. coli from New England Bioworks with our AroGfbr and AroFfbr plasmids, along with our plasmid containing MutMelA (BBa_K2027012, see below).

Melanoma cells share the characteristic of increased melanin production. Accordingly, many treatments have highlighted melanin as an ideal target molecule for radionuclide therapy. By utilizing melanin binding peptides linked to radioactive subunits, melanoma cells can be localized and delivered doses of radiation to eradicate harmful malignant cells. Researchers pursuing this approach to melanoma treatment have made use of phage display libraries to identify multiple small peptides capable of binding melanin. We sought to create a binding domain that would retain large quantities of melanin by engineering a repetitive gene construct containing many copies of the most successful small melanin binding peptides from various studies. Our team decided to pursue the decapeptide known as 4B4 and a hepaptide called 4D, along with irrelevant control peptides PA1 and P601G for reference. [12] [13]

Repetitive 4D and P601G strands were assembled into pSB1C3 backbones with an upstream flanking constitutive promoter and strong RBS. Downstream of the genes, a cellulose binding domain (CBD) extracted from the 2016 distribution kit part BBa_K1321357 via PCR was included as a proof of concept membrane binding domain, separated from the melanin binding module by a GS linker to preserve each domain's function. Following the CBD, we added a FLAG tag, Lumio tag, and His tag for protein presence verification, extraction, and purification. Finally, a double terminator was included before the biobrick suffix to insure termination of transcription.