iGEM UCLA
Spider Silk Genetics Notebook
9/6/2015 - 9/12/2015
9/11/2015 - Protein Purification: Bulk Cell Culture 9/10/2015 - Final Gel Image Pt. 2 9/9/2015 - Final Gel Image 9/8/2015 - Sequencing for M1-15(T7) 9/7/2015 - Colony PCR for M1-15(T7)
8/30/2015 - 9/5/2015
9/4/2015 - Sequencing for Tamura(1C3), Colony PCR for M1-15(T7) 9/3/2015 - Liquid Culture for M1-15(1C3)/(T7) and for Tamura(1C3) 9/2/2015 - Cloning and Transformation for M1-15(1C3)/(T7) 9/1/2015 - Sequencing for M1-12(1C3)/(T7), M1/2[1:2]-12(1C3)/(T7); ICA for M1-15 8/31/2015 - Miniprep and BsaI Digestion for M1-SeqAB2 8/30/2015 - Cloning for M1-12(1C3)/(T7), M1/2[1:2]-12(1C3)/(T7)
8/23/2015 - 8/29/2015
8/28/2015 - Sequencing for M1, M2 monomers; PCR amplification for M1-12, M1/2[1:2]-12 8/27/2015 - Digestion Check for M1/2[1:2]-12(1C3); BsaI Digestion of M1 monomers 8/26/2015 - Sequencing for M1-12(1C3), M1/2[1:1]-12(1C3)/(T7), M1/2[2:1]-12(1C3)/(T7) 8/25/2015 - PCR amplification and cloning for M1/2[1:2]-12 8/24/2015 - Cloning M1-12(1C3), M1/2[1:1]-12(1C3)/(T7), M1/2[2:1]-12(1C3)/(T7); ICA for M1/2[1:2]-12
8/16/2015 - 8/22/2015
8/21/2015 - ICA for M1/2[2:1]-12 8/20/2015 - M1-12(1C3) Size Verification; Maxiprep for M2-AB, BC, CA; BsaI Digestion of M2 Monomers 8/19/2015 - M1-12(1C3) Sequencing Results; ICA for M1/2[1:1]-12 8/18/2015 - Sequencing for M1-12(1C3) 8/17/2015 - Colony PCR, Liquid Culture for M1-12(1C3)
8/9/2015 - 8/15/2015
8/14/2015 - Sequencing for M1-9(T7); PCR amplification for M1-12 8/13/2015 - Colony PCR for M1-SeqAB1 and M1-9(T7); PCR amplification for M1-12 8/12/2015 - Colony PCR for M1-SeqAB 1 and 2 and M1-9(T7); BsaI Digestion for M1 monomers 8/11/2015 - BsaI Digestion for M2 monomers; ICA for 15-mer; Transformation of M1-12(1C3) into DH5(alpha) 8/10/2015 - PCR amplification, EcoRI and PstI digestion for M1-12
8/2/2015 - 8/8/2015
8/7/2015 - PCR for M1-12; BsaI digestion of M1-AB, BC, CA; Digestion for M1-Seq(1/2)AB; Sub-cloning M1-9(1C3) 8/6/2015 - Sequencing results for M2-15(T7), M1-9(1C3) 8/5/2015 - Maxiprep for M1 ICA pieces; Sequencing for M2-15(T7), M1-9(1C3) 8/4/2015 - PCR for M1-12; End extension for M1-Seq1AB, M1-Seq2AB; Colony PCR for M2-15(T7) , M1-9(1C3) 8/3/2015 - PCA for M1-Seq Cores; Digestion, Ligation, Transformation for M1-9, M2-12(1C3)
7/26/2015 - 8/1/2015
8/1/2015 - Sequencing Results from M2-12(T7), M2-15(1C3) 7/31/2015 - Sequencing for M2-12(T7), M2-15(1C3) 7/30/2015 - M1-9 ICA, Colony PCR for M2-12(T7), M2-15(1C3) 7/29/2015 - Digestion, Transformation of M2-12(T7), M2-15(1C3) 7/28/2015 - Maxiprep and BsaI Digestion for M1-AB, BC, CA. Sequencing for M2-12(1C3) 7/27/2015 - M2-12(1C3) Colony PCR, M-15 Amplification 7/26/2015 - Transformation of M2-12(1C3) into DH5(alpha)
7/19/2015 - 7/25/2015
7/24/2015 - PCR amplification for M2-15, Troubleshooting Cloning for M2-12(1C3) 7/23/2015 - ICA for M2-15, Miniprep for M1-AB,BC,CA and M2-12(1C3) for Sequencing 7/22/2015 - BsaI Digestion of M2-SeqAB, Midiprep for M2-AB,BC,CA 7/21/2015 - Cloning M2-12(1C3) and MaSp1 AB, BC, CA 7/20/2015 - Cloning M2-9(T7) and M2-12(1C3), MaSp1 PCR End-Extension
7/12/2015 - 7/18/2015
7/18/2015 - Sequencing Results for M2-3(T7), M2-6(T7), and M2-9(1C3) 7/17/2015 - Miniprep, and Sequencing for M2-3(T7), M2-6(T7), and M2-9(1C3) 7/16/2015 - ICA for MaSp2 12-mer, Liquid Culture for MaSp2 3-mer(T7), MaSp2 6-mer(T7), and MaSp2 9-mer(pSB1C3) 7/15/2015 - Subcloning MaSp2 3-, 6-mer into T7 expression Vector, Cloning MaSp2 9-mer into pSB1C3 7/14/2015 - ICA 9-mer Again: Electric Boogaloo 7/13/2015 - ICA 6-, 9-mer PCR amplification Temperature Testing
7/5/2015 - 7/11/2015
7/10/2015 - ICA for 6-mer and 9-mer 7/9/2015 - Ligation and Transformation of 3-mer, 6-mer and MaSp2 Seq AB 7/8/2015 - EcoRI, PstI Digestion of 3-mer, 6-mer, MaSp2 Seq AB, and pSB1C3: Cloning Preparation 7/7/2015 - Assembly of MaSp2 Sequencing Monomer AB, and ICA testing using M-270 Beads 7/6/2015 - Assembly of MaSp2 Sequencing Core, and ICA testing using M-270 Beads
6/28/2015 - 7/4/2015
7/2/2015 - 6-mer Amplification using Post-elution Primers 7/1/2015 - 6-mer Ligation without beads
6/21/2015 - 6/27/2015
6/26/2015 - ICA Troubleshooting with reduced initiator 6/25/2015 - ICA Troubleshooting to assess bead amplification 6/24/2015 - ICA Troubleshooting to assess bead binding: results 6/23/2015 - ICA Troubleshooting to assess bead binding
6/14/2015 - 6/20/2015
6/19/2015 - Initial ICA Testing 6/18/2015 - BsaI Digestion of VF/R Amplified MaSp2 6/17/2015 - PCR amplification of MaSp using VF2, VR
5/31/2015 - 6/6/2015
6/5/2015 - ICA Oligos Characterization 6/3/2015 - ICA Oligos Characterization 6/2/2015 - Gel purification of BsaI digested MaSp2 6/1/2015 - Preparing to test Initiator, Terminator, and Capping Oligos: PCR amplification and BsaI Digestion
5/24/2015 - 5/30/2015
5/29/2015 - T7 Ligation Test for VF/R amplified MaSp 5/28/2015 - BsaI Digestion of VF/R amplified MaSp2 5/27/2015 - Temperature Test for VF/R amplification of MaSp, Large-scale amplification of MaSp 2AB, 2BC, 2CA 5/26/2015 - BsaI Digestion of VF/R amplified MaSp2
5/17/2015 - 5/23/2015
5/22/2015 - PCR amplification of MaSp2 using VF2, VR 5/21/2015 - PCR amplification of MaSp2 using VF2, VR Cycle Testing 5/20/2015 - PCR amplification of MaSp2 using VF2, VR 5/19/2015 - PCR amplification test for MaSp2 plasmid
5/10/2015 - 5/16/2015
5/13/2015 - NotI Concatemer Ligation 5/12/2015 - T4/T7 Ligation Tests continued 5/11/2015 - BsaI Digestion of MaSp2 Plasmids
5/3/2015 - 5/9/2015
5/8/2015 - T4/T7 Ligation Test 5/7/2015 - BsaI Digestion of MaSp plasmids 5/6/2015 - DNA Maxiprep 5/5/2015 - DNA Miniprep 5/4/2015 - BsaI Digestion Test
4/26/2015 - 5/2/2015
5/1/2015 - Primer Exhaustion Calculation 4/30/2015 - Post-elution PCR amplification of MaSp2 plasmids 4/29/2015 - Post-elution PCR amplification of MaSp2 plasmids 4/28/2015 - Post-elution PCR amplification of MaSp2 plasmids 4/27/2015 - Initial ICA Ligation Test
4/19/2015 - 4/25/2015
4/24/2015 - Sending DNA for sequencing 4/23/2015 - Miniprep DNA for sequencing 4/22/2015 - Colony PCR 4/21/2015 - Ligation of MaSp sequences into pSB1C3 and Transformation 4/20/2015 - PCR amplification of pSB1C3 and EcoRI, PstI digestion
4/12/2015 - 4/18/2015
4/17/2015 - Amplification of pSB1C3 4/16/2015 - PCA of MaSp2 Pieces 4/15/2015 - PCA of MaSp2 Pieces 4/14/2015 - PCA of MaSp2 Pieces 4/13/2015 - PCA of MaSp2 Pieces
Goals Our goal is to be able to implement Iterative Capped Assmebly (ICA) for use in designing custom spider silk genes. The spider silk protein has a highly repetitive primary structure, and its properties can be changed by changing the sequence and identities of these repeats. We want to be able to fully control the primary sequence of our silk gene, and develop a quick, efficient method of customizing assembly of the spider-silk genes.
Achievements From 7/14/2015 onwards, we established ICA as a usable technique to create constructs of varying size and content.
We started testing ICA on 6/19/2015
We successfully constructed biobricks that contain our silk genes and the sticky overhangs. 4/24/2015
We proved that our designed sticky ends work as expected, and exhibit specific binding, showing that ICA can work in spider silk genes. 5/8/2015 and 5/12/2015
We have established a protocol generating fragments to be used in ICA.
We successfully characterized the use of ICA oligos with MaSp AB, BC, CA and showed that they could work in ICA! 6/3/2015
We successfully made a 3-mer using the ICA protocol on M-270 Streptavidin Beads! 7/7/2015
Find a list of our designed protocols here ! List of Parts ICA Parts Collection MaSp Assemblies What to accomplish next We are working on optimizing PCR amplification of MaSp and BsaI digestion so as to get the most amount of working monomers for ICA. --DONE! see here We need to characterize our MaSp monomers and how they behave when used with the initiators, terminators, and capping oligos that are also used in ICA. --DONE! 6/3/2015 We are going to start actual ICA soon. --STARTED! see 6/19/2015 We are optimizing ICA to get it to work just right!
Our final goal is to make a several different 15-mers that have different rations of MaSp1 and MaSp2 that we can express and characterize. Raw lab notebook entries 