First day of lab work for the 2024 UAlberta iGEM team. This week, we received our first set of parts and primer, resuspended them and prepped necessary plasmids for our first cloning, Keratin 31 in pET22b+.

This week, we prepped necessary media and competent cells of DH5ɑ for our experiments.

This week, we tested the plates we made and started on our first cloning of Keratin 31 in pET22b+ and prepped plasmids we plan to use for our future clonings.

This week, we confirmed the successful cloning of Keratin 31 in pET22b+ and started on our second cloning of our FRET based Q-sensor into pJUMP 28 with the J23101 promoter and B0034 RBS used by the 2023 UAlberta iGEM team. We also prepped some competent cells of Rosetta gami, BL 21 and K12 [MG1655].

This week, we attempted our first protein purification for Keratin 31 to yield pure protein that can be used for nanobody screen to identify a high binder that can be used in our FRET based Q-sensor. Here we encountered our first hurdle as no keratin was purified through the method we employed.

This week, our IHP team met with time, who made us aware that our Q-sensor has no commercial value and can only be employed in R&D settings, leading to a drastic change in project direction. This week, we also received our KerDZ, pTac RBS 1, pTac RBS 2, pTac RBS 3, pTac RBS 4 gene blocks.

This week, we were still evaluating a new direction for our project, so minimal lab work was performed. All in all, we made media and extracted plasmid for future use.

No lab work was performed this week as we waited on primers to arrive.

This week, we returned to the lab full force with a new direction and started 4 different clonings simultaneously for pTac_RBS 1_sfGFP_pJUMP 24, pTac_RBS 2_sfGFP_pJUMP 24, pTac_RBS 3_sfGFP_pJUMP 24, pTac_RBS 4_sfGFP_pJUMP 24. We confirmed successful clones through sequencing and towards the end of the week, we also started our fifth cloning of J23119_RBS 1_pJUMP 28.

This week, we started by making media/ plates and characterizing the pTac_RBS 1/2/3/4 parts through both growth curves at 30 and 37^oC in DH5a, Rosetta gami, BL 21 and K 12 and sfGFP purification in various condition in DH5a. We also finished our J23119_RBS 1_pJUMP 28 cloning and confirmed success through colony PCR. We finished the week by making Lennox LB for characterization of J23119_RBS 1_pJUMP 28.

This week, we started off by purifying sfGFP from K12 for characterization of pTac_RBS 1_sfGFP_pJUMP 24, pTac_RBS 2_sfGFP_pJUMP 24, pTac_RBS 3_sfGFP_pJUMP 24, pTac_RBS 4_sfGFP_pJUMP 24. We started characterization of J23119_RBS 1_pJUMP 28 in regular and Lenox LB and started the second titration of keratin purification. Towards the end of the week, we started our sixth cloning of pTac_RBS 1_Spider silk_sfGFP_pJUMP 24.

We started off this week by starting our seventh cloning for J23119_RBS 1_KerDZ_JUMP 28 and continued our sixth cloning for spider silk throughout the week.

This week, we started off by making media and plates required for upcoming clonings and characterizations. Towards the end of thai week, we finished our sixth and seventh clonings for pTac_RBS 1_Spider silk_sfGFP_pJUMP 24 and J23119_RBS 1_KerDZ_JUMP 28 and confirmed their success through colony PCRs.

For our final week, we characterized J23119_RBS 1_pJUMP 28, J23119_RBS 1_KerDZ_pJUMP 28, Keratin 31_pET22b+, and pTac_RBS 1_Spider silk_sfGFP_pJUMP 24 by measuring growth in DH5a, Rosetta gami and BL21 for growth curves. We also attempted our third titration of keratin purification after optimizing lysis method and buffer conditions. The SDS-PAGE for this purification showed us keratin in the pellet which gave us more insight into further optimization of keratin purification.