Notebook Overview

Our team has been working hard on our project for many months, and we are proud to share our exciting results with the iGEM community! Here we present a summary of our daily progress and experiments. Starting in the 2013 spring semester, our team learned laboratory techniques and began experiments. Over the summer, the full 10 member team - including one undergraduate student from another university - worked full-time on the project. Currently, during the 2013 fall semester, some members of the team are continuing to invest time and conduct experiments for the project through the Undergraduate Research Opportunities Program at MIT.

Construction and In Vitro Lab Notebook

Summer Tissue Culture Lab Notebook

June 18th, 2013 Single Color Transfection of verified constitutive eBFP, eYFP, and mKate using Lipofectamine. Our goal was to increase our transfection efficiency. Transfected HEK293. (MP, KL, NH)

June 23rd, 2013 Single Color Transfection of verified constitutive eBFP, eYFP, and mKate using Lipofectamine. Our goal was to increase our transfection efficiency. Transfected HEK293. (NH, KL, JP)

June 26th, 2013 Single Color Transfection of verified constitutive eBFP, eYFP, and mKate using Lipofectamine. Our goal was to increase our transfection efficiency. Transfected HEK293. (NH, KL)

June 27th, 2013 Double Color Transfection of HEK293 verified constitutive eBFP, eYFP, and mKate using Lipofectamine. Our goal was to increase our transfection efficiency. (JP,NH, KL)

June 28th, 2013 Transfection of HEK293. Lipofectamine Optimization – We vary cell density, DNA concentration, and the amount of lipo to determine the optimal conditions for transfection. Unfortunately this transfection failed to yield any results. (NH)

June 29th, 2013 Transfection of HEK293. Lipofectamine Optimization – Lipo vs. the number of cells seeded. (KL)

July 1st, 2013 Transfection of HEK293. Prepped samples from 6.28 transfection for FACs and ran FACs. (CL) DNA/Lipo ratio transfection optimization using mKate and single color transfection of eBFP, eYFP, and mKate for basic microscopy. (JP)

July 2nd, 2013 Transfection of HEK293. Dox induction of a single fluorescent protein (Tre_eYFP).

July 3rd, 2013 Transfection of HEK293. Dox Ladder Single Color. Our goal was to create a graph of fluorescence vs. Dox concentration (KL)

July 4th, 2013 Transfection of HEK293 to verify Hef1A_eYFP-miR503 and TRE-t_eYFP-miR503.

July 5th, 2013 Prepped samples from 7.3 transfection for FACs and ran FACs. (CL) Practice transfection of HEK293 single and double colors. (MP, CL)

July 6th, 2013 Prepped samples from 7.4 transfection for FACs and ran FACs. (JP) Second transfection of the dox ladder in HEK293 (KL)

July 7th, 2013 Prepped samples from 7.5 transfection for FACs and ran FACs

July 9th, 2013 Transfected HK293 with Hef1a_Gal4VP16 and an eYFP with UAS target sites to show use of VP16 as an activator (NH)

July 10th, 2013 Single color transfection of Jurkats with LTX (KL). Use siRNA 451 and 503 on eYFP-4x451ts or eYFP-4x503ts in HEK293 to test receiver cell silencing (KL).

July 11th, 2013 Prepped samples from 7.9 transfection for FACs and ran FACs (NH)

July 12th, 2013 Prepped samples from 7.10 transfection for FACs and ran FACs (NH) HEK 293 practice transfection, single and double color (MP). Transfection of Acyl-Tya-GFP construct in HEK293 cells.

July 13th, 2013 Third repeat of HEK2093 Dox ladder transfection. Repeated Gal4-VP16 transfection.

July 14th, 2013 Prepped samples from 7.12 transfection for FACs and ran FACs (MP) Transfection of HEK293 with mKate with intronic miRNA and eYFP with miRNA target sites.

July 15th, 2013 Prepped samples from 7.13 transfection for FACs and ran FACs (CL) HEK293 transfection of traffic light reporter and Cas9 nuclease px335. Transfection of constitutively expressed split venus constructs with and without leucine zippers. (CL)

July 16th, 2013 Prepped samples from 7.14 transfection for FACs and ran FACs (NH). Transfection of HEK293 with Tre_Gal4VP16 with Dox (NH).

July 17th, 2013 Prepped samples from 7.15 transfection for FACs and ran FACs (KL) Repeated transfection of HEK293 with Acyl-Tya-eGFP (KL).

July 19th, 2013 Prepped samples from 7.17 transfection for FACs and ran FACs (KL) Repeated transfection of HEK293 with Acyl-Tya-eGFP (KL). Transfection of Tre_Gasl4VP16 with varying Dox levels.

July 20th, 2013 Single color transfection of Jurkats with LTX (KL)

July 21th, 2013 Prepped samples from 7.19 transfection for FACs and ran FACs (KL) Transfection of Jurkats with Acyl-Tya-eGFP using LTX (KL)

July 22nd, 2013 Prepped samples from 7.20 transfection for FACs and ran FACs (MP) HEK293 transfection with Hef1a_eYFP-503ts and mKate with intronic mi503

July 23rd, 2013 Prepped samples from 7.21 transfection for FACs and ran FACs (NH) Repeated Transfection of HEK293 with mKate with intronic miRNA and eYFP with miRNA target sites. Jurkat LTX Optimization.

July 24th, 2013 Prepped samples from 7.22 transfection for FACs and ran FACs (CL) HEK293 transfection of constitutively expressed split venus at different ratios (CL). Rh-PE staining of Jurkats (HX)

July 26th, 2013 Prepped samples from 7.24 transfection for FACs and ran FACs (CL) July 29th, 2013 Transfection of HEK293 with Ayl-Tya-GFP. Transfection of Jurkats with Neon.

July 30th, 2013 Rh-PE staining of Jurkats with new dye (HX)

August 1st, 2013 Transfection of constitutively Cas-9 split venus fusions without guide RNAs. Transfection of Cas9 nuclease targeting traffic light reporter

August 3rd, 2013 Prepped samples from 8.1 transfection for FACs and ran FACs (CL)

August 4th, 2013 Transfection of Tre_Gasl4VP16 with varying Dox levels. (NH) Repeated Transfection of HEK293 with mKate with intronic miRNA and eYFP with miRNA target sites.

August 6th, 2013 Prepped samples from 8.4 transfection for FACs and ran FACs (NH)

August 7th, 2013 HEK293 transfection testing the Cre-recombinase reporter (Addgene: pRLG3A - pLCMV_ECFP(loxP)(FRT)EYFP)

August 8th, 2013 Repeated Transfection of HEK293 with mKate with intronic miRNA and eYFP with miRNA target sites.

August 8th, 2013 Prepped samples from 8.4 transfection for FACs and ran FACs (HX)

August 14th, 2013 Transfection of LacI repression without miRNA repression.

August 21st, 2013 Transfection of Jurkats with eyfp with miRNA target sites using Neon (NH)

August 22nd, 2013 Transfection of HEK293 with eYFP with miRNA target sites and co-cultured cells with Jurkat exosomes.

August 23st, 2013 Prepped samples from 8.21 transfection for FACs and ran FACs (MP)

August 24th, 2013 Prepped samples from 8.22 transfection for FACs and ran FACs (NH)

August 26th, 2013 Prepped samples from 8.24 transfection for FACs and ran FACs (NH) Repeated Transfection of Jurkats with eyfp with miRNA target sites using Neon (KL)

Summer Tissue Culture Lab Notebook

September 5th, 2013 Transfection of HEK293 with eYFP with miRNA target sites and co-cultured cells with Jurkat exosomes.

September 7th, 2013 Prepped samples from 9.5 transfection for FACs and ran FACs (KL)

September 16th, 2013 Transfection of HEK193. Cas9-Venus fusions with target plasmid and guide RNA (CL)

September 17th, 2013 Transfection of HEK193. Cas9-Venus fusions with target plasmid and guide RNA (CL)

September 18th, 2013 Prepped samples from 9.16 transfection for FACs and ran FACs (CL)

September 19th, 2013 Prepped samples from 9.17 transfection for FACs and ran FACs (CL)

September 20th, 2013 Transfection of HEK193. Cas9-Venus fusions with target plasmid and guide RNA (CL)

September 21st Transfection of HEK293 with eYFP with miRNA target sites and co-cultured cells with Jurkats. (KL)

September 22nd, 2013 Prepped samples from 9.20 transfection for FACs and ran FACs (CL)

September 23rd Prepped samples from 9.21 transfection for FACs and ran FACs (KL)