2015.igem.org:Users
James’ muffins
Talked to Professors Marshall Stark and John Christie about our project.
Repressors
Ordered PPhlF and PSrpR with BioBrick prefix and suffix as oligos. Ordered PhlF and SrpR as G-blocks, with ribosome binding site B0032, terminator B0010+ (a longer version of B0010, that we will characterise and submit as a new BioBrick) and BioBrick prefix and suffix. Got E5501 and I13500 – GFP (E0040) with ribosome binding sites B0032 and B0034 respectively – in pSB1C3 in DH5α from glycerol stocks from last year’s iGEM team.
UVA sensor
Designed primers to PCR UirS and UirR from Synechocystis PCC6803 genomic DNA with ribosome binding site B0032 and BioBrick prefix and suffix. Tested UVB sensitivity of strains TOP10 and DH5α (recA- - very poor DNA repair), DS941 (recF- - limited DNA repair), and MG1655.Z1 (wild type for DNA repair).
Bioluminescence
pBAD.LuxCDABEG (K325909) and pBAD.LuxCDABEG.LumP (K769020) off distribution plates and transformed into TOP10 and DH5α. Designed primers to PCR LuxA, B, C, D, E, and G individually with ribosome binding site B0032 and BioBrick prefix and suffix from K325909.
Vilija’s carrot cake
So good, honestly.
Bioluminescence
Tested K325909 and K769020 in TOP10 to see how long until the bioluminescence started after addition of arabinose, how bright it is, and how LumP affects it.
Repressors
Ligated both PPhlF and PSrpR with E5501 and I13500 in pSB1C3, respectively, and transformed into TOP10 and DH5α. C0040 (TetR), R0040 (PTetR) and backbone pSB3K3 (with insert J04450) off distribution plates – to use C0040 as a control for our repressors, and to use pSB3K3 as a second plasmid to have the promoters and repressors on separate plasmids. Ordered G-Block of R0011.B0032 (PLacI + RBS) for C0040 – as the plasmid on the plate has to RBS.
UVA sensor
Found genomic DNA from Synechocystis PCC6803 to use for PCR. Ordered PLsiR as a G-Block with BioBrick prefix and suffix.
Mhairi’s flapjacks
Talked to Amber Jones from the Glasgow School of Art about our project – changed to nightlight toys.
Bioluminescence
Started PCR of Lux genes from K325909 (twice), only LuxB worked.
Repressors
Ligated R0040 with I13500 and E5501 separately in pSB1C3, R0011.B0032 oligo with C0040 in pSB1C3, and B0032.PhlF.B0010+ and B0032.SrpR.B0010+ G-Blocks separately in pSB1C3. Attempted transformations into TOP10 and DH5α with both electrocompetent cells, and chemically competent cells, but there was no growth, except on the control plates. Repeated this week’s ligations.
UVA sensor
Started PCR of UirR from Synechocystis PCC6803 genomic DNA (twice), didn’t work. Ligated PLsiR into pSB1C3.
Cara’s lemon drizzle cake
Pretty cool,^0^
Bioluminescence
PCR from K325909 worked for LuxA, B, C, D, E and G worked. Ligated into pSB1C3 and transformed into TOP10. No growth.
Repressors
Transformed redone ligations of R0040.I13500, R0040.E5501, R0011.B0032.C0040, B0032.PhlF.B0010+, and B0032.SrpR.B0010+ all in pSB1C3 into TOP10.
UVA sensor
PCR of UirR from Synechocystis PCC6803 genomic DNA worked, ligated into pSB1C3 and transformed into TOP10. PCR of UirS from Synechocystis PCC6803 genomic DNA, ligated into TOPO TA cloning plasmid and transformed into TOP10. Designed primers for site-directed mutagenesis of UirS to remove restriction sites.
Adele’s millionaire shortcake
Feel like I'm millionaire, LOL.
Bioluminescence
Redid PCR of LuxA, B, C, D, E and G from K325909 and gel purified the PCR product before ligating into pSB1C3 and transforming into TOP10. Designed primers for ribosome binding site library.
Repressors
Ligated R0011N with B0032.PhlF.B0010+ and B0032.SrpR.B0010+, separately, and transformed into TOP10.
UVA sensor
Purified oligos for site-directed mutagenesis. Ligated PLsiR in pSB1C3 with E5501 and I13500, and transformed into TOP10.
Adele’s millionaire shortcake
So good, honestly.
Bioluminescence
Redid PCR of LuxA, B, C, D, E and G from K325909 and gel purified the PCR product before ligating into pSB1C3 and transforming into TOP10. Designed primers for ribosome binding site library.
Repressors
Ligated R0011N with B0032.PhlF.B0010+ and B0032.SrpR.B0010+, separately, and transformed into TOP10.
UVA sensor
Purified oligos for site-directed mutagenesis. Ligated PLsiR in pSB1C3 with E5501 and I13500, and transformed into TOP10.
Adele’s millionaire shortcake
So good, honestly.
Bioluminescence
Redid PCR of LuxA, B, C, D, E and G from K325909 and gel purified the PCR product before ligating into pSB1C3 and transforming into TOP10. Designed primers for ribosome binding site library.
Repressors
Ligated R0011N with B0032.PhlF.B0010+ and B0032.SrpR.B0010+, separately, and transformed into TOP10.
UVA sensor
Purified oligos for site-directed mutagenesis. Ligated PLsiR in pSB1C3 with E5501 and I13500, and transformed into TOP10.
Adele’s millionaire shortcake
So good, honestly.
Bioluminescence
Redid PCR of LuxA, B, C, D, E and G from K325909 and gel purified the PCR product before ligating into pSB1C3 and transforming into TOP10. Designed primers for ribosome binding site library.
Repressors
Ligated R0011N with B0032.PhlF.B0010+ and B0032.SrpR.B0010+, separately, and transformed into TOP10.
UVA sensor
Purified oligos for site-directed mutagenesis. Ligated PLsiR in pSB1C3 with E5501 and I13500, and transformed into TOP10.
Adele’s millionaire shortcake
So good, honestly.
Bioluminescence
Redid PCR of LuxA, B, C, D, E and G from K325909 and gel purified the PCR product before ligating into pSB1C3 and transforming into TOP10. Designed primers for ribosome binding site library.
Repressors
Ligated R0011N with B0032.PhlF.B0010+ and B0032.SrpR.B0010+, separately, and transformed into TOP10.
UVA sensor
Purified oligos for site-directed mutagenesis. Ligated PLsiR in pSB1C3 with E5501 and I13500, and transformed into TOP10.
Adele’s millionaire shortcake
So good, honestly.
Bioluminescence
Redid PCR of LuxA, B, C, D, E and G from K325909 and gel purified the PCR product before ligating into pSB1C3 and transforming into TOP10. Designed primers for ribosome binding site library.
Repressors
Ligated R0011N with B0032.PhlF.B0010+ and B0032.SrpR.B0010+, separately, and transformed into TOP10.
UVA sensor
Purified oligos for site-directed mutagenesis. Ligated PLsiR in pSB1C3 with E5501 and I13500, and transformed into TOP10.
Adele’s millionaire shortcake
So good, honestly.
Bioluminescence
Redid PCR of LuxA, B, C, D, E and G from K325909 and gel purified the PCR product before ligating into pSB1C3 and transforming into TOP10. Designed primers for ribosome binding site library.
Repressors
Ligated R0011N with B0032.PhlF.B0010+ and B0032.SrpR.B0010+, separately, and transformed into TOP10.
UVA sensor
Purified oligos for site-directed mutagenesis. Ligated PLsiR in pSB1C3 with E5501 and I13500, and transformed into TOP10.
Adele’s millionaire shortcake
So good, honestly.
Bioluminescence
Redid PCR of LuxA, B, C, D, E and G from K325909 and gel purified the PCR product before ligating into pSB1C3 and transforming into TOP10. Designed primers for ribosome binding site library.
Repressors
Ligated R0011N with B0032.PhlF.B0010+ and B0032.SrpR.B0010+, separately, and transformed into TOP10.
UVA sensor
Purified oligos for site-directed mutagenesis. Ligated PLsiR in pSB1C3 with E5501 and I13500, and transformed into TOP10.