Team:Grinnell/Notebook/Gels/DspB

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<img alt ='20110708_dspB%2BPrsaA_Pxyl' src='https://static.igem.org/mediawiki/2011/b/bc/20110708_dspB%2BPrsaA_Pxyl.jpg' />
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<img alt ='20110708_dspB%2BpromotersVSdspB_std' src='https://static.igem.org/mediawiki/2011/1/17/20110708_dspB%2BpromotersVSdspB_std.jpg' />
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Gel proving the successful transformation of WT <i>dspB</i> from UBC into <i>E. coli</i> Top10. Lane 1: ladder; lanes 2-4: digests (EcoRI and PstI) of miniprep DNA from overnights of transformant colonies 1-3. There is a clear band at the appropriate size for <i>dspB</i> in all of the experimental lanes.
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Figure 1: Gel proving the successful transformation of WT <i>dspB</i> from UBC into <i>E. coli</i> Top10. Lane 1: ladder; lanes 2-4: digests (EcoRI and PstI) of miniprep DNA from overnights of transformant colonies 1-3. There is a clear band at the appropriate size for <i>dspB</i> in all of the experimental lanes.
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Gel showing <i>dspB</i> with various promoter inserts as contrasted to a standard <i>dspB</i> with no promoter. Lane 1: dspB (WT) + P<sub>rsaA</sub>; lane 2: with P<sub>xyl</sub>; lanes 3,4: with BBa_K081005; lane 5: standard <i>dspB</i>; lane 6: ladder. Not all transformants show successful insertion of the desired promoter, but it is clear which were successful when compared to the standard <i>dspB</i>.
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Figure 2: Gel of transformants that should contain both WT <i>dspB</i> and a promoter insert. Lane 1: ladder; lanes 2-4: WT <i>dspB</i> with P<sub>rsaA</sub> colonies 1-4; lanes 5-7: WT <i>dspB</i> with P<sub>xyl</sub> colonies 1-4; lane 8: standard <i>dspB</i> with no promoter.
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Figure 3: Follow up to Figure 2.  Gel showing WT <i>dspB</i> with various promoter inserts as contrasted to a standard <i>dspB</i> with no promoter. Lane 1: WT <i>dspB</i> + P<sub>rsaA</sub> colony 3; lane 2: with P<sub>xyl</sub> colony 3; lanes 3,4: with BBa_K081005 colonies 3 and 4; lane 5: standard WT <i>dspB</i>; lane 6: ladder.
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Gel of transformants that should contain both <i>dspB</i> and a promoter insert. Lane 1: ladder; lanes 2-4: <i>dspB</i> with P<sub>rsaA</sub>; lanes 5-7: <i>dspB</i> with P<sub>xyl</sub>; lane 8: standard <i>dspB</i> with no promoter.
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Figure 4: Test of more WT <i>dspB</i> with P<sub>xyl</sub> colonies. Lane 1: ladder; lanes 2-5: WT <i>dspB</i> with P<sub>xyl</sub> colonies 5-8; lane 6: standard <i>dspB</i> with no promoter.
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Gel of more transformants of <i>dspB</i> with P<sub>xyl</sub>. Lane 1: ladder; lanes 2-5: transformants; lane 6: standard <i>dspB</i> with no promoter.
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Gel of transformants with synthesized <i>dspB</i> and <i>esp</i> with codons optimized for expression in <i>Caulobacter</i> after ligation into pSB1C3. Lane 1: ladder; lanes 2-5: optimized <i>dspB</i>; lanes 6-9: optimized <i>esp</i>. Lane 4 shows successful insertion of <i>dspB</i> into pSB1C3, but none of the optimized <i>esp</i> transformants show the desired insert.
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Figure 5: Optimized <i>dspB</i> transformed into pSB1C3 colonies A-E. Lane 1: ladder; lanes 2-5: optimized <i>dspB</i> colonies A-E; lanes 6-9: optimized <i>esp</i>. Colony D shows the expected size.
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Gel of colony PCR of transformants that should carry WT <i>dspB</i> behind the P<sub>xyl</sub> promoter. Lanes 1-4: colonies 9, 10, 12, and 15 from transformation plate; lane 5: <i>dspB</i> WT standard from PCR.
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Figure 6: More colonies of WT <i>dspB</i> with P<sub>xyl</sub> promoter. Lanes 1-4: colonies 9, 10, 12, and 15; lane 5: <i>dspB</i> WT standard from PCR.
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Gel for extraction of WT <i>dspB</i> behind P<sub>xyl</sub>. Lane 1: ladder; lanes 2,3: miniprep digest.
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Figure 7: The rest of the PCR product of colony 10 from Figure 6 in preparation for gel extraction of WT <i>dspB</i> behind P<sub>xyl</sub>. Lane 1: ladder; lanes 2,3: WT <i>dspB</i> with P<sub>xyl</sub>.
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Gel of colony PCR of transformants carrying optimized <i>dspB</i> and <i>rsaA</i> C-terminal. Lane 1: ladder; lanes 2-5: transformation colonies 1-4; lane 6: <i>dspB</i> standard from PCR of WT <i>dspB</i>.
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Figure 8: Gel of colony PCR of transformants carrying optimized <i>dspB</i> and <i>rsaA</i> C-terminal. Lane 1: ladder; lanes 2-5: transformation colonies 1-4; lane 6: <i>dspB</i> standard from PCR of WT <i>dspB</i>.
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Lane 1: ladder; lanes 2-5: transformants that should carry P<sub>rsaA</sub>, optimized <i>dspB</i>, and <i>rsaA</i>; lanes 6-9: transformants that should carry P<sub>xyl</sub>, optimized <i>dspB</i>, and <i>rsaA</i>; lane 10: standard optimized <I>dspB</I> from PCR. None of these transformants showed success.
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Figure 9: Optimized <i>dspB</i> + <i>rsaA</i> with P<sub>xyl</sub> and P<sub>rsaA</sub>.  Lane 1: ladder; lanes 2-5: P<sub>rsaA</sub> + optimized <i>dspB</i> + <i>rsaA</i> colonies 1-4; lanes 6-9: P<sub>xyl</sub> + optimized <i>dspB</i> + <i>rsaA</i> colonis 1-4; lane 10: standard optimized <I>dspB</I> + <i>rsaA</i>. P<sub>rsaA</sub> + optimized <i>dspB</i> + <i>rsaA</i> colony 3 shows the expected size.
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Lane 1: ladder; lanes 2-5: transformants that should carry P<sub>xyl</sub>, optimized <I>dspB</i>, and <I>rsaA</i>; lane 6: standard optimized <i>dspB</i> with <i>rsaA</i> from PCR. None of these colonies were successful.
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Figure 10: Colonies 5-8 of P<sub>xyl</sub> + <I>dspB</i> + <I>rsaA</i>. Lane1: ladder; lanes 2-5: carry P<sub>xyl</sub> + optimized <I>dspB</i> + <I>rsaA</i>; lane 6: standard optimized <i>dspB</i> with <i>rsaA</i> from PCR. None of these colonies were successful.
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Lane 1: standard optimized <i>dspB</i> with <i>rsaA</i> from PCR; lanes 2,3: transformants that should carry WT <i>dspB</i> behind P<sub>xyl</sub>; lane 4: ladder. No successful transformations.
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Figure 11: Lane 1: standard optimized <i>dspB</i> with <i>rsaA</i> from PCR; lanes 2,3: transformants that should carry WT <i>dspB</i> behind P<sub>xyl</sub>; lane 4: ladder. No successful transformations.
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Lane 1: ladder; lanes 2-5: transformants that should carry BBa_K081005, optimized <i>dspB</i>, and <i>rsaA</i>; lane 6: standard optimized <i>dspB</i> with <i>rsaA</i> fragment. Lanes 3 and 5 carry the desired combination of genes.
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Figure 12: BBa_K081005 + <I>dspB</i> + <I>rsaA</i> colonies 1-4.  Lane 1: ladder; lanes 2-5: BBa_K081005 + optimized <i>dspB</i> + <i>rsaA</i>; lane 6: standard optimized <i>dspB</i> with <i>rsaA</i> fragment. Colony 2 is successful.
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Lane 1: ladder; lanes 2-7: transformants that should carry P<sub>xyl</sub>, optimized <i>dspB</i>, and <i>rsaA</i>; lane 8: standard fragment of optimized <i>dspB</i> with <i>rsaA</i>. None of the transformants carried the desired gene sequence.
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Figure 13: P<sub>xyl</sub> + <I>dspB</i> + <I>rsaA</i> colonies 9-14. Lane 1: ladder; lanes 2-7: P<sub>xyl</sub> + <I>dspB</i> + <I>rsaA</i>; lane 8: standard fragment of optimized <i>dspB</i> with <i>rsaA</i>.
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Lane 1: ladder; lanes 2-5: transformants that should carry BBa_K081005, optimized <I>dspB</i>, and <i>rsaA</i> in pMR10; lanes 6-9: transformants that should carry P<sub>rsaA</sub>, optimized <I>dspB</i>, and <i>rsaA</i> in pMR10; lane 10: standard DNA fragment of optimized <i>dspB</i> with <i>rsaA</i>. Lane 7 carries the desired gene sequence.
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Figure 14: Lane 1: ladder; lanes 2-5: BBa_K081005, optimized <I>dspB</i>, and <i>rsaA</i> in pMR10; lanes 6-9: P<sub>rsaA</sub>, optimized <I>dspB</i>, and <i>rsaA</i> in pMR10; lane 10: standard DNA fragment of optimized <i>dspB</i> with <i>rsaA</i>. çolony 2 carries P<sub>rsaA</sub> + <I>dspB</i> + <i>rsaA</i>.
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Gel of PCR of transformants that should carry P<sub>xyl</sub>, optimized <i>dspB</i>, and <i>rsaA</i>. Lane 1: ladder; lanes 2-6: PCR products; lane 7: standard fragment of <i>dspB</i> with <i>rsaA</i>.
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Figure 15: Gel of PCR of transformants that should carry P<sub>xyl</sub>, optimized <i>dspB</i>, and <i>rsaA</i>. Lane 1: ladder; lanes 2-6: PCR products; lane 7: standard fragment of <i>dspB</i> with <i>rsaA</i>.
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Gel of PCR of transformants that should carry P<sub>xyl</sub>, optimized <i>dspB</i>, and <i>rsaA</i>. Lane 1: ladder; lanes 2-9: transformant PCR products; lane 10: digested miniprep of what should have been WT <i>dspB</i>, but showed up as the wrong fragment size.
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Figure 16: Gel of PCR of transformants that should carry P<sub>xyl</sub>, optimized <i>dspB</i>, and <i>rsaA</i>. Lane 1: ladder; lanes 2-9: transformant PCR products; lane 10: digested miniprep of what should have been WT <i>dspB</i>, but showed up as the wrong fragment size.
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PCR of transformants that should carry BBa_K081005, optimized <i>dspB</i>, and <i>rsaA</i> in pMR10. Lane 1: ladder; lanes 2-7: PCR products.
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Figure 17: PCR of transformants that should carry BBa_K081005, optimized <i>dspB</i>, and <i>rsaA</i> in pMR10. Lane 1: ladder; lanes 2-7: PCR products.
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PCR of transformants that should carry P<sub>xyl</sub>, optimized <i>dspB</i>, and <i>rsaA</i> in pMR10. Lane 1: ladder; lanes 2-8: PCR products.
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Figure 18: PCR of transformants that should carry P<sub>xyl</sub>, optimized <i>dspB</i>, and <i>rsaA</i> in pMR10. Lane 1: ladder; lanes 2-8: PCR products.
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==2011.07.24-2011.07.31==
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Figure 19: Lane 1: ladder; lanes 2-9: colony PCR of transformants that should contain BBa_K081005, optimized <i>dspB</i>, and <i>rsaA</i>.
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Latest revision as of 14:10, 5 August 2011

Grinnell Menubar

Gels for DspB

2011.07.03-2011.07.09

20110707_UBC_dspB_transformation_results_colonies_1_2_3 20110708_dspB%2BPrsaA_Pxyl 20110708_dspB%2BpromotersVSdspB_std 20110709dspB%2Bpxyl_Colonies5-8
Figure 1: Gel proving the successful transformation of WT dspB from UBC into E. coli Top10. Lane 1: ladder; lanes 2-4: digests (EcoRI and PstI) of miniprep DNA from overnights of transformant colonies 1-3. There is a clear band at the appropriate size for dspB in all of the experimental lanes. Figure 2: Gel of transformants that should contain both WT dspB and a promoter insert. Lane 1: ladder; lanes 2-4: WT dspB with PrsaA colonies 1-4; lanes 5-7: WT dspB with Pxyl colonies 1-4; lane 8: standard dspB with no promoter. Figure 3: Follow up to Figure 2. Gel showing WT dspB with various promoter inserts as contrasted to a standard dspB with no promoter. Lane 1: WT dspB + PrsaA colony 3; lane 2: with Pxyl colony 3; lanes 3,4: with BBa_K081005 colonies 3 and 4; lane 5: standard WT dspB; lane 6: ladder. Figure 4: Test of more WT dspB with Pxyl colonies. Lane 1: ladder; lanes 2-5: WT dspB with Pxyl colonies 5-8; lane 6: standard dspB with no promoter.


2011.07.10-2011.07.16

20110710_optGenes%2BC3 20110711_c9_10_12_15_from20110707transf-Pxyl%2BdspBWT 20110712_dspB%2BPxyl_forGX 20110712_optdspB%2BrsaAc1-4_optdspB
Figure 5: Optimized dspB transformed into pSB1C3 colonies A-E. Lane 1: ladder; lanes 2-5: optimized dspB colonies A-E; lanes 6-9: optimized esp. Colony D shows the expected size. Figure 6: More colonies of WT dspB with Pxyl promoter. Lanes 1-4: colonies 9, 10, 12, and 15; lane 5: dspB WT standard from PCR. Figure 7: The rest of the PCR product of colony 10 from Figure 6 in preparation for gel extraction of WT dspB behind Pxyl. Lane 1: ladder; lanes 2,3: WT dspB with Pxyl. Figure 8: Gel of colony PCR of transformants carrying optimized dspB and rsaA C-terminal. Lane 1: ladder; lanes 2-5: transformation colonies 1-4; lane 6: dspB standard from PCR of WT dspB.
20110714_optdspB%2BrsaA%2BPrsaA_OR_Pxyl 20110714_optdspB%2BrsaA%2BPxyl_c5-8 20110714_WTdspB%2BPxyl 20110715_BBa%2BoptdspB%2BrsaA
Figure 9: Optimized dspB + rsaA with Pxyl and PrsaA. Lane 1: ladder; lanes 2-5: PrsaA + optimized dspB + rsaA colonies 1-4; lanes 6-9: Pxyl + optimized dspB + rsaA colonis 1-4; lane 10: standard optimized dspB + rsaA. PrsaA + optimized dspB + rsaA colony 3 shows the expected size. Figure 10: Colonies 5-8 of Pxyl + dspB + rsaA. Lane1: ladder; lanes 2-5: carry Pxyl + optimized dspB + rsaA; lane 6: standard optimized dspB with rsaA from PCR. None of these colonies were successful. Figure 11: Lane 1: standard optimized dspB with rsaA from PCR; lanes 2,3: transformants that should carry WT dspB behind Pxyl; lane 4: ladder. No successful transformations. Figure 12: BBa_K081005 + dspB + rsaA colonies 1-4. Lane 1: ladder; lanes 2-5: BBa_K081005 + optimized dspB + rsaA; lane 6: standard optimized dspB with rsaA fragment. Colony 2 is successful.
20110715_Pxyl%2BoptdspB%2BrsaA_c9-14
Figure 13: Pxyl + dspB + rsaA colonies 9-14. Lane 1: ladder; lanes 2-7: Pxyl + dspB + rsaA; lane 8: standard fragment of optimized dspB with rsaA.

2011.07.17-2011.07.23

20110718_BBa_OR_PrsaA%2BoptdspB%2BrsaA_pMR10 20110719_Pxyl%2BodspB%2BrsaA_re-do_c1,3,4,5,6_std 20110720_Pxyl%2BodspB%2BrsaA_re-do_c7-14_WTdspB_c5 20110722_BBa%2BodspB%2BrsaA%2BpMR10_c1,5-9
Figure 14: Lane 1: ladder; lanes 2-5: BBa_K081005, optimized dspB, and rsaA in pMR10; lanes 6-9: PrsaA, optimized dspB, and rsaA in pMR10; lane 10: standard DNA fragment of optimized dspB with rsaA. çolony 2 carries PrsaA + dspB + rsaA. Figure 15: Gel of PCR of transformants that should carry Pxyl, optimized dspB, and rsaA. Lane 1: ladder; lanes 2-6: PCR products; lane 7: standard fragment of dspB with rsaA. Figure 16: Gel of PCR of transformants that should carry Pxyl, optimized dspB, and rsaA. Lane 1: ladder; lanes 2-9: transformant PCR products; lane 10: digested miniprep of what should have been WT dspB, but showed up as the wrong fragment size. Figure 17: PCR of transformants that should carry BBa_K081005, optimized dspB, and rsaA in pMR10. Lane 1: ladder; lanes 2-7: PCR products.
20110722_Pxyl%2BodspB%2BrsaA%2BpMR10
Figure 18: PCR of transformants that should carry Pxyl, optimized dspB, and rsaA in pMR10. Lane 1: ladder; lanes 2-8: PCR products.

2011.07.24-2011.07.31

20110725_BBa%2BodspB%2BrsaA_c10-17
Figure 19: Lane 1: ladder; lanes 2-9: colony PCR of transformants that should contain BBa_K081005, optimized dspB, and rsaA.

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