Difference between revisions of "Team:Glasgow/Notebook"
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| − | <h1><strong>Repressors</strong></h1><p>Ordered | + | <h1><strong>Repressors</strong></h1><p>Ordered P<sub><i>PhlF</i></sub> and P<sub><i>SrpR</i></sub> 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) with 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. </p> |
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| − | <h1><strong>UVA sensor</strong></h1><p>Designed primers to PCR | + | <h1><strong>UVA sensor</strong></h1><p>Designed primers to PCR <i>uirS</i>, <i>uirR</i>, and P<sub><i>lsiR</i></sub> from <i>Synechocystis</i> PCC6803 genomic DNA with ribosome binding site B0032 and BioBrick prefix/suffix. Tested UVB sensitivity of strains TOP10 and DH5α (<i>recA<sup>-</sup></i> mutants - very poor DNA repair), DS941 (<i>recF<sup>-</sup></i> - limited DNA repair), and MG1655.Z1 (wild type for DNA repair).</p> |
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| − | <h1><strong>Bioluminescence</strong></h1><p>pBAD.LuxCDABEG (K325909) and pBAD.LuxCDABEG.LumP (K769020) | + | <h1><strong>Bioluminescence</strong></h1><p>Retrieved plasmid DNA of pBAD.LuxCDABEG (Cambridge Luxbrick: K325909) and pBAD.LuxCDABEG.LumP (K769020) from 2015 iGEM distribution plates and transformed into TOP10 and DH5α strains. Designed primers to PCR LuxA, B, C, D, E, and G individually with ribosome binding site B0032 and BioBrick prefix/suffix from K325909. </p> |
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| − | <h1><strong>Bioluminescence</strong></h1><p>Tested K325909 and K769020 in TOP10 to see how long until | + | <h1><strong>Bioluminescence</strong></h1><p>Tested K325909 and K769020 in TOP10 to see how long until there is expression of bioluminescence after addition of arabinose inducer, how bright the bioluminescence is, and how LumP affects it in K769020.</p> |
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| − | <h1><strong>Repressors</strong></h1><p>Ligated both | + | <h1><strong>Repressors</strong></h1><p>Ligated both P<i><sub>PhlF</sub></i> and P<i><sub>SrpR</sub></i> with E5501 and I13500 in pSB1C3, respectively, and transformed into TOP10 and DH5α. Retrieved C0040 (TetR), R0040 (P<i><sub>TetR</sub></i>) 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 (P<i><sub>LacI</sub></i> + RBS) for C0040 – as the plasmid on the plate has to RBS.</p> |
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| − | <h1><strong>UVA sensor</strong></h1><p>Found genomic DNA from Synechocystis PCC6803 to use for PCR. Ordered | + | <h1><strong>UVA sensor</strong></h1><p>Found genomic DNA from <i>Synechocystis</i> PCC6803 to use for PCR. Ordered P<sub><i>LsiR</i></sub> as a G-Block with BioBrick prefix and suffix.</p> |
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| − | <h1><strong>UVA sensor</strong></h1><p> | + | <h1><strong>UVA sensor</strong></h1><p>Found <i>Synechocystis</i> PCC6803 genomic DNA in Sean's freezer. Started PCR of <i>uirR</I> and <i>uirS</I> (twice), didn’t work. Ligated P<sub><I>LsiR</i></sub> G-Block into pSB1C3.</p> |
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| − | <h1><strong>UVA sensor</strong></h1><p>PCR of | + | <h1><strong>UVA sensor</strong></h1><p>PCR of <i>uirS</i> and <i>uirR</i> from Synechocystis PCC6803 genomic DNA worked, ligated <i>uirR</i> into pSB1C3 and transformed into TOP10. PCR of <i>uirS</i> was ligated into TOPO TA cloning plasmid and transformed into TOP10. Designed primers for site-directed mutagenesis of <i>uirS</i> to remove incompatible restriction sites.</p> |
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| − | <h1><strong>UVA sensor</strong></h1><p>Purified oligos for site-directed mutagenesis. Ligated | + | <h1><strong>UVA sensor</strong></h1><p>Purified oligos for site-directed mutagenesis. Ligated P<sub><i>LsiR</i></sub> in pSB1C3 with E5501 and I13500, and transformed into TOP10.</p> |
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| − | <h1><strong> | + | <h1><strong>.</p> |
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| − | <h1><strong>Repressors</strong></h1><p> | + | <h1><strong>Repressors</strong></h1><p>.</p> |
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| − | <h1><strong>UVA sensor</strong></h1><p> | + | <h1><strong>UVA sensor</strong></h1><p>.</p> |
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| − | <h1><strong>Bioluminescence</strong></h1><p> | + | <h1><strong>Bioluminescence</strong></h1><p>.</p> |
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| − | <h1><strong>Repressors</strong></h1><p> | + | <h1><strong>Repressors</strong></h1><p>.</p> |
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<li>3rd PCR mutagenesis of UirS – so hopefully fixed, needs to be sequenced</li> | <li>3rd PCR mutagenesis of UirS – so hopefully fixed, needs to be sequenced</li> | ||
<li>Chromophore (PCB) BioBricks as miniprep and checked by digest (has a constitutive promoter)</li> | <li>Chromophore (PCB) BioBricks as miniprep and checked by digest (has a constitutive promoter)</li> | ||
| − | <li> | + | <li>Confirmed P<sub><i>LsiR</i></sub> is off when not activated (tested by GFP with growth curve)</li> |
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Revision as of 21:31, 14 November 2015
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) with 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, uirR, and PlsiR from Synechocystis PCC6803 genomic DNA with ribosome binding site B0032 and BioBrick prefix/suffix. Tested UVB sensitivity of strains TOP10 and DH5α (recA- mutants - very poor DNA repair), DS941 (recF- - limited DNA repair), and MG1655.Z1 (wild type for DNA repair).
Bioluminescence
Retrieved plasmid DNA of pBAD.LuxCDABEG (Cambridge Luxbrick: K325909) and pBAD.LuxCDABEG.LumP (K769020) from 2015 iGEM distribution plates and transformed into TOP10 and DH5α strains. Designed primers to PCR LuxA, B, C, D, E, and G individually with ribosome binding site B0032 and BioBrick prefix/suffix from K325909.
Vilija’s carrot cake
So good, honestly.
Bioluminescence
Tested K325909 and K769020 in TOP10 to see how long until there is expression of bioluminescence after addition of arabinose inducer, how bright the bioluminescence is, and how LumP affects it in K769020.
Repressors
Ligated both PPhlF and PSrpR with E5501 and I13500 in pSB1C3, respectively, and transformed into TOP10 and DH5α. Retrieved 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
Found Synechocystis PCC6803 genomic DNA in Sean's freezer. Started PCR of uirR and uirS (twice), didn’t work. Ligated PLsiR G-Block 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 uirS and uirR from Synechocystis PCC6803 genomic DNA worked, ligated uirR into pSB1C3 and transformed into TOP10. PCR of uirS was ligated into TOPO TA cloning plasmid and transformed into TOP10. Designed primers for site-directed mutagenesis of uirS to remove incompatible 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.
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Repressors
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UVA sensor
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Bioluminescence
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Repressors
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UVA sensor
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Ye’s cakes
Bioluminescence
- Ligating promoter (pBAD) upstream of LuxAB – test by spraying with decanal
- Transformed LuxCD into DH5a and DS941.Z1 – ligate in R0011N (will be repressed in those strains); ligate Lux E downstream
- checking if intermediates of tetradecanal synthesis is toxic to cells (hence a repressed promoter)
- Checking sequenced of Lux genes with mutations (different from genomic Vibrio sequence) – if both colonies are the same, may be mutations in luxoperon (K325909) – need to design primers
- Ribosome binding site library
- PCR products of LuxA-G individually, transforming into
- LuxA ligated into pBAD-pSB1C3
- LuxC ligated into R0011N-pSB1C3
- LuxB,D,E, and G ligated into pSB1C3 with no promoter
- Ligate pBAD-(library)-LuxA with (library)-LuxB and spray with decanal
Repressors
- Have promoters+GFP in pSB3K3 in DS941.Z1
- Have R0011N+repressors in pSB1C3 in DS941.Z1 – make competent cells, and transform in promoters+GFP in pSB3K3
- Growth curve and fluorescence to test promoters
- Bi-stable switch: have PPhlF+SrpR, PSrpR+PhlF in pSB1C3 – need to make and check miniprep, then transform promoters+GFP in pSB3K3
- Make one promoter with GFP, one with RFP on same plasmid (one needs terminator)
- Terminators: ligate “steve” (B0010+), B0010, and B0015 into pSB1A10
UVA sensor
- 3rd PCR mutagenesis of UirS – so hopefully fixed, needs to be sequenced
- Chromophore (PCB) BioBricks as miniprep and checked by digest (has a constitutive promoter)
- Confirmed PLsiR is off when not activated (tested by GFP with growth curve)
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