Team:Concordia/Demonstrate/Recombinant

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SYNTHESIS ATTACHMENT MICROFLUIDICS CDC28, FHUA-GBP, AND MELA IMPROVEMENT

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Overview

As the name International Genetically Engineered Machine describes, iGEM is a competition that harnesses the use of molecular and genetic techniques for innovation and design of novel tools and devices for real-world applications. In order to harmonize the newly emerging field of nanotechnology with genetic engineering, iGEM Concordia’s 2016 team has been experimenting with effective methods of nanoparticle synthesis and attachment to different cell types, including Escherichia coli bacteria and Saccharomyces cerevisiae yeast. In order to work toward nanoparticle synthesis and attachment, molecular cloning methods were utilized, and several BioBricks parts were improved in the process. iGEM Concordia has successfully contributed parts: BBa_K2045000 and BBa_K2045003 to the iGEM parts registry.


BBa_K2045001 FhuA-GBP: Ferrichrome Iron outer membrane transporter-gold binding peptide
In order to effectively bind gold nanoparticles to E.coli, Ferrichrome Iron outer membrane transporter was used as a binding intermediate between the E.coli cell and gold nanoparticles. This was done by improving iGEM BCCS-Bristol’s part, BBa_K259000, by fusing a gold binding peptide with their part which was FhuA. We sought to synthesize and display the gold nanoparticles on the surface of E.coli cells. A gold binding peptide is a series of amino acids that have an affinity to gold, mostly sulfide based amino acids, since sulfide groups have a natural chemical affinity to gold. This property makes it a vital part for iGEM Concordia for effective attachment of gold nanoparticles onto the cell surface of E.coli. In order to accomplish this; gold nanoparticles were displayed by fusing the gold binding peptide with FhuA. FhuA is a ferrichrome Iron outer membrane transporter which we fused GBP with to create FhuA-GBP. The binding of gold nanoparticles occurred in cells expressing FhuA-GBP. These findings demonstrate the effectiveness of the proposed method in the synthesis and display of gold nanoparticles.This part has many future applications and will allow for the attachment of gold nanoparticles to many kinds of protein structures and will be a future key player in the emerging use of nanotechnology in synthetic biology.


BBa_K2045002 CDC28: Cyclin-dependent kinase catalytic subunit
In order to have effective cell battle with our microgladiators… It was important to ensure that cell division of our nanoparticle coated cells didn’t occur. Cell division would not only make it difficult for cell isolation in our microfluidic battle dome, but it will also compromise the integrity of the cell’s nanoweaponry; since the coating of nanoparticles will be halved as the cell membrane goes through cell division. The solution was CDC28, a gene that allows users to control the growth of their cells by steadily controlled external temperature. The cells will only be active and capable of cell division at 24oC and inactive, therefore incapable of cell division, at 37oC. By attaching nanoparticles to our yeast cells with CDC28, and keeping them at 37oC in the microfluidic battle system, we can ensure an effective battle between our micro-gladiators without compromising their attached nanoweapons. Since the presence of the wild type CDC28 gene in the yeast genome would mask the phenotype of the temperature sensitive mutant we decided to knock out the wild type version of the gene using CrispR. We designed 2 primers that would amplify the T.S. CDC28 with 3 silent mutations. CrispR gRNAs were then designed to bind to the wild type sequence of the gene but were unable to bind to the newly mutated version of the T.S. CDC28 due to the 3 silent mutations. The gRNAs and the mutated T.S. CDC28 were introduced to the wild type yeast and were selected for by antibiotic resistance. Future iGEM teams will be able to use our primer and gRNA designs in order to turn wild type yeast into cell cycle temperature sensitive yeast mutants.

1- PCR amplify temperature sensitive CDC28 gene with these primers
Forward primer: 5’ caaccgttaggagcAgaCatCgttaag 3’
Reverse primer: 5’ ctttactagActGtaCtgacagtgcagtagc 3’
(silent mutations in upper case and bolded)
2-Use newly amplified T.S. CDC28 as a donor our 2 guide RNA constructs for CrispR chain reaction
gRNA1: gctgatattgttaagaagtt
gRNA2: tataatgacagtgcagtagc


To characterize this biobrick, we grew an overnight culture of the temperature-sensitive CDC28 mutant and wild-type yeast cells. After diluting the cell solutions to an optical density of 0.1, both strains were grown in SC media at 24°C and 37°C in microplates. The OD at 600nm was measured every 20 minutes for 24 hours using a Tecan Sunrise machine to create growth curves for both the wild-type and the CDC28 mutant at the two temperatures. The results clearly indicate that at the nonpermissive temperature of 37°C there is a significant difference between the growth of the wild-type and mutant. The CDC28 mutant strain stays at a low OD while the wild-type displays a typical growth curve. At the permissive temperature of 24°C, there is little difference in the pattern of growth between the two cell strains. This confirms the ability of our biobrick part to inhibit growth at a specific temperature of 37°C.

BBa_K274001 MelA Cambridge
iGEM Concordia has showcased the synthesis of nanoparticles through many chemical and plant based methods. iGEM Concordia wanted to find an innovative application where we will be able to develop a system where we can synthesize and attach nanoparticles through a single biological cell system.
MelA is a gene that produces Eumelanin, which is a dark pigment. Eumelanin has carboxyl and hydroxyl quinone groups, whose charges help reduce gold ions, present in the cytoplasm, into gold nanoparticles. In order to accomplish this, we used iGEM Cambridge’s part, BBa_K274001, for nanoparticle production. The characterization of Cambridge’s part was incomplete therefore we conducted extensive characterization of the melA gene. To do this we performed to assays on the melA gene. the first being a time course in order to screen for melanin production in timed intervals. In the second assay, tyrosine concentrations will be varied, the large sample effect will optimize characterization of the MelA part.(characterization).In order to make more gold nanoparticles within the E.coli cell, iGEM Concordia IMPROVED the part in order to have higher expression of MelA for higher gold nanoparticle synthesis.This is done by a point mutation, (Santos CNS, Stephanopoulos G., 2007).


During sequence verification of the plasmid construct, a mutant was discovered containing a C→T base pair substitution at the 1,000th nucleotide of the melA gene, a change that results in a proline-to-serine switch in the 334th amino acid. The heightened expression of melA, part BBa_K2045004,caused by a point mutation. showed signs of melanin synthesis 12 h ahead of the wild type. A pTac promoter and a T1 terminator was added in order to regulate melA induction in controlled environments for a controlled synthesis of gold nanoparticles within bacteria.