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Team:Bielefeld-CeBiTec/Notebook/Journal/rMFC/Jul
From 2014.igem.org
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<ul> | <ul> | ||
| - | <li><b> oprF </b></li> | + | <li><b> deletion of <i>dcuB</i> and integration of <i>oprF</i> into chromosome </b></li> |
<ul> | <ul> | ||
| - | <li><a href=" | + | <li> This week we started the deletion of the fumarate/succinate antiporter <i>dcuB</i> and the integration of outer membrane porin <i> oprF</i> into chromosome using the <a href="http://www.genebridges.com/storage/Manuals_PDF/K006%20Ecoli%20Gene%20Deletion%20Kit-version2.3-2012.pdf" target="_blank">Genebridge Red/ET-System</a> |
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<ul> | <ul> | ||
| - | <li> | + | <li>pRedET plasmid</li> |
<ul> | <ul> | ||
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<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pRedET plasmid </li> | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pRedET plasmid </li> | ||
| + | </ul> | ||
| + | <li> oprF (<a href="http://parts.igem.org/wiki/index.php/Part:http://parts.igem.org/wiki/index.php/Part:BBa_K1172507" target="_blank">BBa_K1172507</a>)</li> | ||
| + | <ul> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1C3_BBa_K1172507 </li> | ||
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</ul> | </ul> | ||
| + | </ul> | ||
</ul> | </ul> | ||
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<ul> | <ul> | ||
| - | <li><b> | + | <li><b> deletion of <i>dcuB</i> and integration of <i>oprF</i> into chromosome</b></li> |
| - | <ul> <li> This week we | + | <ul> <li> This week we amplified the <i>pR6K</i>-cassette and the <i>oprF</i> gene to connect them to each other. Additionally we transform the RedET plasmid into the <i> E. coli </i> strain KRX. |
<ul> | <ul> | ||
<li><i><b>pR6K</b></i> | <li><i><b>pR6K</b></i> | ||
Revision as of 16:13, 5 October 2014
 |
July |
 |
- Amplification of pSB1C3 backbone for FumA and FumBCD
- This week we amplified the pSB1C3 backbone of FumA and FumBCD to use it for Gibson Assembly afterwards
- PCR amplification ( pSB1C3_pre_Fum_A, pSB1C3_suf_Fum_A )
- Annealing temperature: 55 °C
- Bands are as expected (2,07 kb)
- PCR amplification ( pSB1C3_pre_Fum_BCD, pSB1C3_suf_Fum_BCD )
- Annealing temperature: 55 °C
- Bands are as expected (2,07 kb)
- PCR purification of backbones
- deletion of dcuB and integration of oprF into chromosome
- This week we started the deletion of the fumarate/succinate antiporter dcuB and the integration of outer membrane porin oprF into chromosome using the Genebridge Red/ET-System
- pRedET plasmid
- Plasmid isolation of pRedET plasmid
- oprF (BBa_K1172507)
- Plasmid isolation of pSB1C3_BBa_K1172507
- deletion of dcuB and integration of oprF into chromosome
- This week we amplified the pR6K-cassette and the oprF gene to connect them to each other. Additionally we transform the RedET plasmid into the E. coli strain KRX.
- pR6K
- PCR amplification of pR6K-cassette (pR6K_dcuB_fwd, pR6K_dcuB_rev)
- Annealing temperature: 55 °C
- Bands are as expected (~1600 bp) but slightly visible
- PCR amplification of pR6K-cassette is repeated two times
- PCR product pR6K was purified out of the gel
- oprF
- PCR amplification of oprF for integration into chromosome ( porine_fwd_FRT, porine_rev_FRT )
- Annealing temperature: 55 °C
- bands are as expected (~1359 bp)
- PCR product oprF was purified out of the gel
- connection of pR6K and oprF
- PCR amplification to connect the pR6K-cassette and oprF (porine_fwd_FRT, pR6K_dcuB_rev)
- Annealing temperature: 55 °C
- Bands not as expected because of too much template (~2900 bp)
- Next time the PCR amplification was planned with a different usage of primer
- additionally a new PCR amplification (as described further up) of pR6K-cassette and a new purification of it and oprF out of the gel was done
- PCR amplification to connect the pR6K-cassette and oprF was repeated with the new purified pR6K-cassette and oprF and different primer usage
- PCR amplification was initially performed 15 cycles without primer and afterwards 30 cycles with addition of primer
- RedET plasmid
- Transformation with electrocompotetent cells of the RedET plasmid into E. coli KRX
- pR6K
- connection of pR6K-cassette and oprF
- PCR amplification to connect the pR6K-cassette and oprF
- PCR amplification and purification of pR6K-cassette and oprF seperatly
