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Gel 1 of 2. Lane 1: ladder; lanes 2-4: PCR product using VF2 and VR of plasmid containing esp ligated with rsaA C-terminal that had been digested 20110606; lane 5: PCR product of plasmid containing esp; lanes 6-8: PCR product using VF2 and VR of plasmid containing esp ligated with rsaA C-terminal PCR product from 20110605. Only lane 6 shows successful ligation of esp and rsaA C-terminal.
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Gel 2 of 2. Lane 1: ladder; lanes 2-4: PCR product using VF2 and VR of plasmid containing esp ligated with rsaA C-terminal that had been digested 20110606; lane 5: PCR product of plasmid containing esp; lanes 6-8: PCR product using VF2 and VR of plasmid containing esp ligated with rsaA C-terminal PCR product from 20110605. None of these show successful ligation.
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Gel of PCR products of transformations of insertion of Biobrick promoter BBa_K081005 into pSB1C3 containing esp and rsaA C-terminal. Lane 1: ladder; lanes 2-4 and 6-8: PCR product of transformation survivors; lane 5: control DNA of esp + rsaA C-terminal. Only lane 2 shows something that may be success, but as the fragment for the promoter is so small, it is difficult to confirm based solely on gel electrophoresis.
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Gel of PCR product of esp and rsaA C-terminal using new primers. Lane 1: ladder; lanes 2,3: PCR product of rsaA from chromosomal Caulobacter DNA; lane 4: PCR product of esp using new primers from chromosomal S. epidermidis DNA.
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Lanes 1-6: plasmid PCR of transformed cells that should carry the desired esp insert; lane 7: ladder; lanes 8-10: plasmid PCR of transformed cells that should be carrying esp + rsaA C-terminal insert. A few of the esp inserts succeeded, but as expected the three piece ligation failed again.
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Lanes 1-3 and 5-8: plasmid PCR of transformed cells that should carry the Promoter-esp-stop-rsaA insert in plasmid pMR10; lane 4: ladder. Only lane 3 shows any success.
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Lanes 1-3 and 5-7: plasmid PCR of colonies that should be carrying rsaA C-terminal insert in pSB1C3; lane 4: ladder. Lanes 3 and 4 seem to show insert at significant concentrations.
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Gel 1 of 2. Colony PCR of transformation products of insertions of esp from PCR into pSB1C3 containing rsaA C-terminal, insertions of rsaA C-terminal into pSB1C3 containing esp, and esp and rsaA C-terminal digests from pSB1C3 into pMR10 as a three piece ligation. Lane 1: ladder; lanes 2-4: esp from PCR inserted into rsaA containing pSB1C3; lanes 5-7: rsaA from PCR inserted into esp containing pSB1C3; lanes 8-10: esp and rsaA digests from plasmid insertion into pMR10. Lanes 2-4 show correct insertion length, but none of the other fragments are the correct length.
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Gel 2 of 2. Colony PCR of transformation products of insertions of esp from PCR into pSB1C3 containing rsaA C-terminal, insertions of rsaA C-terminal into pSB1C3 containing esp, and esp and rsaA C-terminal digests from pSB1C3 into pMR10 as a three piece ligation. Lane 1: ladder; lanes 2-4: esp from PCR inserted into rsaA containing pSB1C3; lanes 5-7: rsaA from PCR inserted into esp containing pSB1C3; lanes 8-10: esp and rsaA digests from plasmid insertion into pMR10. Lanes 2-4 show correct insertion length, but none of the other fragments are the correct length.
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Colony PCR products of transformation products of insertions of esp + rsaA C-terminal into plasmid containing Caulobacter constitutive promoter PrsaA. Lane 1: ladder; lanes 2-7: PCR products; lane 8: standard PCR product of pSB1C3 containing only PrsaA. Transformation was successful, but ligation, and perhaps digestion, were not.
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Colony PCR products of transformation products of insertion of esp + rsaA into pSB1C3 containing Caulobacter inducible promoter Pxyl (induced in presence of xylene). Lane 1: ladder; lanes 2-7: PCR products; lane 8: standard PCR product of pSB1C3 containing only Pxyl. Transformation was successful, but ligation was not.
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Colony PCR amplified DNA fragments of transformation products of esp + rsaA inserts into plasmid containing existing Biobrick promoter BBa_K081005. Lane 1: ladder; lanes 2-7: PCR products; lane 8: standard DNA fragment identified as containing BBa_K081005. The lack of DNA may be due to a breakdown in our antibiotic; the transformation is being repeated.
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Gel 1 of 2. PCR products of transformation cells that should contain esp + rsaA C-terminal insert into plasmids containing various promoters. Lane 1: ladder; lanes 2-5: inserts into plasmid containing BBa_K081005; lane 6: standard BBa_K081005 containing no insert; lanes 7-10: inserts into plasmid containing Pxyl. While the BBa_K081005 plasmids seem to have some insert, none of these show an insert of appropriate size (~1.3-1.5kb).
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Gel 2 of 2. PCR products of transformation cells that should contain esp + rsaA C-terminal insert into plasmids containing various promoters. Lane 1: ladder; lanes 2-7: inserts into plasmid containing PrsaA; lane 8: standard PrsaA with no insert; lane 9: standard Pxyl with no insert. The first lane shows odd results, but none of these seems to have the desired insert.
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Lane 1: ladder; lane 2: colony PCR product for promoter BBa_K081005 in preparation for digestion and insertion into plasmid containing esp and rsaA C-terminal. Result is positive.
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Gel 1 of 3. PCR of transformation cells that should contain an insert of Pxyl into pSB1C3 containing esp and rsaA C-terminal. Lane 1: ladder; lanes 2-5: PCR with plasmid primers VF2 and VR; lanes 6-9: PCR with promoter specific forward primer and plasmid reverse primer VR. The lack of DNA is disturbing and suggests that our plates are ineffective or contaminated.
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Gel 2 of 3. PCR of transformation cells that should contain an insert of PrsaA into pSB1C3 containing esp and rsaA C-terminal. Lane 1: ladder; lanes 2-5: PCR with plasmid primers VF2 and VR; lanes 6-9: PCR with promoter specific forward primer and plasmid reverse primer VR. Bands in lanes 2, 3, and 4 suggests that these colonies at least contained the plasmid, but the apparent size of the band is too small for esp and rsaA C-terminal together; rather it is the correct size for just rsaA C-terminal.
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Gel 3 of 3. PCR product of transformation colonies that should contain an insert of either Pxyl or PrsaA into pSB1C3 containing esp and rsaA C-terminal. Lane 1: ladder; lanes 2,3: Pxyl insert PCR using plasmid primers VF2 and VR; lanes 4,5: PrsaA insert PCR using plasmid primers VF2 and VR; lanes 6,7: Pxyl insert PCR using promoter specific forward primer and VR; lanes 8,9: PrsaA insert PCR using promoter specific forward primer and VR. Lanes 8 and 9 suggest a successful insertion of PrsaA, however all of the bands are too small.
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Verification of presence of lack of rsaA C-terminal insert in pSB1C3 containing esp. Lane 1: ladder; lane 2: digested PCR of possible insert; lane 3: standard digested esp. The band is faint, but there appears to be a band at the same height as the standard as well as a couple higher bands for leftover plasmids, suggesting that there was no insertion of rsaA C-terminal.
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Gel 1 of 2. PCR of transformation products of rsaA C-terminal insert into pSB1C3 containing esp. Lane 1: ladder; lanes 2-7: ligation 1, colonies 1-6; lanes 8-10: ligation 2, colonies 1-3. The complete lack of DNA fragments here means that these ligations failed.
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Gel 2 of 2. PCR of transformation products of rsaA C-terminal insert into pSB1C3 containing esp. Lane 1: ladder; lanes 2-4: ligation 2, colonies 4-6; lanes 5-10: ligation 3, colonies 1-6. All the bands here are correct for esp, but not the combination.
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PCR test of transformation products of insertion of gel extracted DNA into plasmid containing another gene. Lane 1: ladder; lanes 2-4: insertion of rsaA C-terminal into plasmid containing esp; lanes 5-7: insertion of esp into plasmid containing rsaA C-terminal. Smearing of bands is inconclusive and suggests that there may be no DNA (colonies are contaminants) or that freeze-thaw did not succeed in providing sufficient template DNA for PCR.
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Digested miniprep samples of overnight cultures of transformation product that may contain esp and rsaA. Lane 1: esp standard; lanes 2,3: rsaA from gel extraction inserted into pSB1C3 containing esp from plates spread with 20μL of transformation cells; lanes 4,5: rsaA from gel extraction inserted into pSB1C3 containing esp from plates spread with 200μL of transformation cells; lanes 6,7: esp from gel extraction inserted into pSB1C3 containing rsaA from plates spread with 20μL of transformation cells; lanes 8,9: esp from gel extraction inserted into pSB1C3 containing rsaA from plates spread with 200μL of transformation cells; lane 10: ladder.
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Lane 1: ladder; lane 2: digest of miniprep of transformation product of insert of esp from gel extraction into plasmid containing rsaA; lanes 3,4: ligations gel extracted esp and rsaA and plasmid containing the other gene; lane 5: ligation of gel extracted esp with rsaA. Lane 2 shows that ligation was unsuccessful. The rest of the lanes are inconclusive and a follow up gel with additional DNA is to be run.
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Lane 1: ladder; lane 2: ligation of esp and rsaA C-terminal fragments from gel extraction. No rsaA appears to be present, nor any ligation, only esp.
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Gel of digested pSB1C3 containing esp for gel extraction. Lane 1: ladder; lanes 2,3: digest with EcoRI and SpeI; lanes 5,6: digest with SpeI and PstI.
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Gel of digests of pSB1C3 containing rsaA C-terminal in preparation for gel extraction. Lane 1: ladder; lanes 3,4: digest with XbaI and PstI; lanes 6,7: digest with EcoRI and XbaI.
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Gel of restriction digests (EcoRI and PstI) of miniprepped DNA from more transformants from the weekend's transformations.
Lane 1: ladder; lanes 2-5: colonies that should contain rsaA inserted into pSB1C3 containing esp; lanes 6-9: colonies that should contain esp inserted into pSB1C3 containing rsaA; lane 10: esp standard.
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Follow up gel of lanes 5 and 6 from the previous gel, redigested for a longer period of time. Lane 1: ladder; lane 2: digest of miniprep product from colony that should contain an insert of rsaA into plasmid already containing esp (previous lane 5); lane 3: digest of miniprep product from colony that should contain an insert of esp into plasmid already containing rsaA (previous lane 6).
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Gel for gel extraction of esp and rsaA combination and checking the identity of PCR product. Lane 1: ladder; lanes 3,4: digest of miniprep DNA for gel extraction; lane 6: PCR product. Gel is somewhat messy, but appropriately sized bands are apparent.
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Gel of transformation products of esp + rsaA insert into plasmid containing promoter BBa_K081005. Lane 1: ladder; lanes 2-5: transformants using gel extracted combo gene; lanes 6-9: transformants using PCR product for combo insert.
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Gel of transformants that should carry esp + rsaA behind a promoter. Lane 1: ladder; lanes 2-5: PrsaA with insert; lanes 6-9: Pxyl with insert. None of these transformants show the desired insert.
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Gel of more transformants that should have esp + rsaA combo inserted behind Pxyl. Lane 1: ladder; lanes 2-4: combo from miniprep digest and gel extract insert; lanes 5-7: combo from PCR insert; lane 8: confirmation of insertion behind BBa_K081005; lane 9: standard combo from gel extraction.
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Gel of transformants that should carry esp + rsaA combo insert behind PrsaA run against standard combo. Lane 1: ladder; lanes 2-4: combo from miniprep digest and gel extract insert; lanes 5-7: combo from PCR product insert; lane 8: standard combo without any promoter.
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Gel of colony PCR of transformants that should carry BBa_K081005, WT esp, and rsaA in pMR10, Pxyl, WT esp, and rsaA in pSB1C3, or PrsaA, WT esp, and rsaA in pSB1C3. Lane 1: ladder; lanes 2-5: BBa_K081005, WT esp, and rsaA in pMR10; lanes 6,7: Pxyl, WT esp, and rsaA in pSB1C3; lanes 8,9: PrsaA, WT esp, and rsaA in pSB1C3.
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Lane 1: ladder; lanes 2-5: colony PCR of transformants that should carry the optimized esp in pSB1C3; lane 6: follow up digest of miniprep of transformant that may carry optimized esp in IDTSMART-AMP. None of these colonies were successful.
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Lane 1: ladder; lanes 2-9: transformants that should carry optimized esp in IDTSMART-AMP; lane 10: confirmation of digest of miniprep of strain carrying BBa_K081005, WT esp, and rsaA in pMR10. Lanes 4, 7, and 9 show successful transformation.
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Lane 1: ladder; lanes 2-4: transformants that should carry optimized esp in pSB1C3; lanes 5-10: transformants that should carry optimized esp behind PrsaA. Lane 9 has the desired gene sequence.
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Lane 1: ladder; lanes 2-4: transformants that should carry optimized esp in pSB1C3; lanes 5-10: transformants that should carry optimized esp behind Pxyl. Lanes 5 and 7 have the desired gene sequence.
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Lane 1: ladder; lanes 2-7: transformants that should carry optimized esp with rsaA from gel extraction in IDTSMART-AMP. Lanes 3, 4, and 6 have the desired gene sequence.
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PCR of transformants that should carry PrsaA, optimized esp, and rsaA or optimized esp with rsaA in pSB1C3. Lane 1: ladder; lanes 2-7:combo with PrsaA; lanes 8-10: combo without promoter. Lanes 3, 8, and 10 all have the desired inserts.
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Lane 1: ladder; lanes 2-7: PCR of transformants that should carry Pxyl, optimized esp, and rsaA; lanes 8-10: transformants that should carry optimized esp with rsaA in pSB1C3.
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Lane 1: ladder; lanes 2-7: transformants that should carry BBa_K081005 and optimized esp.
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Gel of digests of miniprepped overnight liquid culture of cells that should carry BBa_K081005 and optimized esp. Lane 1: ladder; lanes 2-10: digests.
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Lane 1: ladder; lanes 2-7: PCR of transformants that should carry PrsaA, optimized esp, and rsaA in pMR10. Lane 4 was successful.
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Lane 1: ladder; lanes 2-7: PCR of transformants that should carry Pxyl, optimized esp, and rsaA in pMR10.
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Lane 1: ladder; lanes 2-8: digests of transformants that should contain Pxyl, optimized esp, and rsaA.
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Lane 1: ladder; lanes 2-8: digests of miniprep of transformants that should carry BBa_K081005, optimized esp, and rsaA. No success.
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Lane 1: ladder; lanes 2-5: PCR of Caulobacter colonies believed to carry BBa_K081005, WT esp, and rsaA. All passed.
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Lane 1: ladder; lanes 2,3: PCR of transformants that should carry BBa_K081005, optimized esp, and rsaA in pMR10; lanes 4-10: PCR of transformants that should carry BBa_K081005, optimized esp, and rsaA in pSB1C3. No success.
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