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| | [[Edinburgh/Notebook/Plates|Plates]]<br /> | | [[Edinburgh/Notebook/Plates|Plates]]<br /> |
| | [[Edinburgh/Notebook/Gels|Gels]]<br /> | | [[Edinburgh/Notebook/Gels|Gels]]<br /> |
| - |
| |
| - | '''P1:''' ''dxs'' from ''Eschaerichia coli'' JM109 (or K12?)<br />
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| - | '''P2:''' ''appY'' from ''E. coli'' JM109 (or K12?)<br />
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| - | '''P3:''' ''glgC'' from ''E. coli'' JM109 (or K12?)<br />
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| - | '''P4:''' fusion PCR for BABEL1+''glgC''<br />
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| - | '''P5:''' fusion PCR for BABEL2+''glgC''<br />
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| - | '''P6:''' mutagenic PCR to remove EcoRI site 1 from ''glgC''<br />
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| - | '''P7:''' mutagenic PCR to remove EcoRI site 2 from ''glgC''<br />
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| - | '''P8:''' ''crtB'' (from ''Pantoea ananatis'' cells)<br />
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| - | '''P9:''' ''crtI'' (from Douglas's maxiprep of the mutated ''crtIB'' plasmid)<br />
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| - | '''P10:''' ''crtY'' (from ''P. ananatis'' cells). Failed.<br />
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| - | '''P11:''' fusion PCR for rbs+''dxs''. Failed.<br />
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| - | '''P12:''' repeat fusion PCR for rbs+''dxs'', using L11 and primers rbs2fclon1+pSB1A2insr1.<br />
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| - | '''P13 and P14:''' MABEL mutation of site 1 on ''glgC'' mutant clones M19 and M22.<br />
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| - | '''P15:''' fusion PCR for rbs+''appY''<br />
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| - | '''P16, P17, P18 and P19:''' first attempts at ''cenA'', ''cenB'', ''cenC'' and ''cex''.<br />
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| - | '''P20, P21, P22 and P23:''' second attempts at ''cenA'', ''cenB'', ''cenC'' and ''cex''.<br />
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| - | '''P24, P25:''' repeat PCR for ''cenA'' and ''cex'' using Pfu. Failed.<br />
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| - | '''P26:''' Positive control PCR with primers fd1 and rd1 to amplify 16S rRNA gene ''rrnB'' from ''Cellulomonas fimi''.<br />
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| - | '''P27:''' ''crtY'' from ''P. ananatis'' cells. - Failed<br />
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| - | '''P28, P29:''' ''crtB'' and ''crtI'' from L18 and L19.<br />
| |
| - | '''P30:''' Repeat positive control ''rrnB'' from ''C. fimi'', annealing 1 minute.<br />
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| - | '''P31:''' pZntA promoter<br />
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| - | '''P32-P35:''' repeat PCR for ''cenA'', ''cenB'', ''cenC'' and ''cex'', annealing 1 minute. Failed.<br />
| |
| - | '''P36:''' M43 (''glgC''-mut1,2) with primers glgCf2/glgCr2 (25.07.08: AM)<br />
| |
| - | '''P37:''' ''crtY'' from ''P. ananatis''. (25.07.08: AM) - Failed<br />
| |
| - | '''P38~P41:''' ''cenA'', ''cenB'', ''cenC'' and ''cex'' from heat-killed cell suspension.<br />
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| - | '''P42~P45:''' ''cenA'', ''cenB'', ''cenC'' and ''cex'' from 'impure' DNA solution.<br />
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| - | '''P46~P47:''' ''cenA'' and ''cex'' from 'impure' DNA solution, annealing 65C. Failed.<br />
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| - | '''P48:''' ''crtY'' (new primer mixture) (previously P38a) (30.07.08: CF) - Failed<br />
| |
| - | '''P49:''' Recreation of P12 (rbs+''dxs'') (previously P39a) (30.07.08: CF)<br />
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| - | '''P50:''' third mutagenesis of ''glgc''-mut1,2 (04.08.08: YAN)<br />
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| - | '''P51:''' pCstA from ''E. coli'' cells (06.08.08: CF) - Failed<br />
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| - | '''P52:''' ''cenA'' from heat killed ''C. fimi'' (07/08/08: YAN, AM) - Failed<br />
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| - | '''P53:''' ''cex'' from heat killed ''C. fimi'' (07/08/08: YAN, AM) - Failed<br />
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| - | '''P54:''' pCstA from ''E. coli'' cells - Purified (07.08.08: CF)<br />
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| - |
| |
| - | ==== Ligations ====
| |
| - |
| |
| - | '''L1:''' Edinbrick1+''dxs''<br />
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| - | '''L2:''' Edinbrick1+''appY''<br />
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| - | '''L3:''' Babel1+''glgC''<br />
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| - | '''L4:''' Babel2+''glgC''<br />
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| - | '''L5:''' Edinbrick1+''appY'' repeat (initially labelled L4 in error)<br />
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| - | '''L6:''' Self ligation of mutagenic PCR P6<br />
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| - | '''L7:''' Self ligation of mutagenic PCR P7<br />
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| - | '''L8:''' Ligation of synthetic ribosome binding site to M2 (BBa_K118000)<br />
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| - | '''L9:''' Edinbrick1+''crtB''<br />
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| - | '''L10:''' Edinbrick1+''crtI'' (mutant lacking PstI sites, I hope)<br />
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| - | '''L11:''' repeat ligation of synthetic ribosome binding site to M2 (BBa_K118000)<br />
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| - | '''L12:''' rbs ligated with ''appY''.<br />
| |
| - | '''L13 and L14:''' self ligations of ''glgC'' mutation reactions P13 and P14.<br />
| |
| - | '''L15:''' ligation of Edinbrick1 with rbs+''dxs'' fusion PCR product (P12). - Failed<br />
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| - | '''L16:''' Ligation of Edinbrick1 with rbs+''appY'' fusion PCR product (P15) (17/07/08) - Failed<br />
| |
| - | '''L17:''' Ligation of ''crtB'' (M36) to rbs (22/07/08)<br />
| |
| - | '''L18:''' Ligation of ''crtI'' (M42) to rbs (22/07/08)<br />
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| - | '''L19:''' pSB1A2+rbs+''dxs'' ligation product (From P12) (25/07/08: Yan/OG)<br />
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| - | '''L20:''' pSB1A2+rbs+''appY'' ligation product (From P15) (25/07/08: Yan/OG)<br />
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| - | '''L21:''' pSB1A2+rbs+''crtB'' ligation product (From P28) (25/07/08: Yan/OG)<br />
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| - | '''L22:''' pSB1A2+rbs+''crtI'' ligation product (From P29) (25/07/08: Yan/OG)<br />
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| - | '''L23:''' PzntA+pSB1A2 ligation product (From P31) (25/07/08: Yan/OG)<br />
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| - | '''L24:''' Ligation of ''crtE'' to pSB1A2 (25/07/08: CF)<br />
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| - | '''L25:''' Ligation of ''glgC'' double mutant (P36) to Edinbrick 1 (28.07.2008: AM)<br />
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| - | '''L26:''' Ligation of Edi+P15 XP - rbs+''appY'' (30.07.2008: HX)<br />
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| - | '''L27:''' Ligation of Edi+P28 XP - rbs+''crtB'' (30.07.2008: HX)<br />
| |
| - | '''L28:''' Putative ''cenA'' ligated to Edinbrick1 (30.07.08: AM)<br />
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| - | '''L29:''' Putative ''cex'' ligated to Edinbrick1 (30.07.08: AM)<br />
| |
| - | '''L30:''' Self-ligation of P15 (3rd mutagenesis of ''glgC''-mut1,2) (05.08.08: HX)<br />
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| - | '''L31:''' Ligation of P36 (''glgC''-mut1,2, hopefully, could be P39) to Edinbrick1 (05.08.08: OG)<br />
| |
| - | '''L32:''' Ligation of rbs+''crtB'' (M67) to rbs+''crtI'' (M50) to create ''crtB''/''crtI'' (07.08.08: AM, Yan)<br />
| |
| - | '''L33:''' Ligation of PcstA (P54) to Edinbrick1 (08.08.08: OG)<br />
| |
| - |
| |
| - | ==== Minipreps ====
| |
| - |
| |
| - | '''M1 to M6:''' pSB1A2+''dxs'' transformants. *M2, M3, M4 and M6* all looked correct on a gel. *M2* was sequenced to confirm identity and was maxiprepped as *X2*.<br />
| |
| - | '''M7:''' Babel1+''glgC'' transformant. Did not look right on gel.<br />
| |
| - | '''M8 to M12:''' Babel2+''glgC''_ transformants. *M10 and M11* both looked good on a gel, but sequencing showed that both had the insert in the reverse orientation.<br />
| |
| - | '''M13 to M18:''' pSB1A2+''appY'' transformants. Gel 10 showed that M15 to M18, and possibly M14, all seemed to have inserts the right size.<br />
| |
| - | '''M19 to M24:''' Babel2+''glgC'' clones after mutation of EcoRI site 2. EcoRI digests on *Gel 11* suggest that *M19, M21 and M22* may have lost the EcoRI site.<br />
| |
| - | '''M25 to M30:''' possible rbs+''dxs'' transformants.<br />
| |
| - | '''M31 to M36:''' ''crtB'' clones.<br />
| |
| - | '''M37 to M42:''' ''crtI'' clones.<br />
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| - | '''M43 to M47:''' possible ''glgC'' double mutants (both EcoRI sites gone)<br />
| |
| - | '''M48:''' a possible rbs+''dxs'' clone.<br />
| |
| - | '''M49 to M54:''' pSB1A2-rbs+''crtI''<br />
| |
| - | '''M55 to M60:''' PZntA promoter<br />
| |
| - | '''M61 to M66:''' BABEL2+rbs+''crtE''<br />
| |
| - | '''M67:''' rbs+''crtB''<br />
| |
| - | '''M68 to M71:''' pSB1A2+''glgC''-mut1,2 (31.07.08: AM, YAN)<br />
| |
| - | '''M72 to M75:''' pSB1A2+rbs+''dxs'' (31.07.08: AM, YAN)<br />
| |
| - | '''M76 to M79:''' BABEL2+''crtE'' (31.07.08: AM, YAN)<br />
| |
| - | '''M80 to M83:''' pSB1A2+rbs+''appY'' (04/08/08: YAN and OG)<br />
| |
| - | '''M84 to M85:''' pSB1A2+rbs+''crtB'' (04/08/08: YAN and OG)<br />
| |
| - | '''M86 to M90:''' pSB1A2+''cenA'' (04/08/08: YAN and OG)<br />
| |
| - | '''M91 to M96:''' pSB1A2+''cex''(04/08/08: YAN and OG)<br />
| |
| - | '''M97 to M104:''' pSB1A2+''cenA'' (05.08.08: YAN)<br />
| |
| - | '''M105 to M108:''' pSB1A2+''cex'' (05.08.08: YAN)<br />
| |
| | | | |
| | ==== Maxipreps ==== | | ==== Maxipreps ==== |
"
