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Team:Bielefeld-CeBiTec/Notebook/Journal/rMFC/Jul
From 2014.igem.org
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| - | <li><b> | + | <li><b> Deletion of <i>dcuB</i> and integration of <i>oprF</i> into chromosome </b></li> |
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<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> | <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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| - | <li><b> | + | <li><b> Deletion of <i>dcuB</i> and integration of <i>oprF</i> into chromosome</b></li> |
<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> <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. | ||
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| + | <a href="https://static.igem.org/mediawiki/2014/3/33/Bielefeld_CeBiTec_2014-09-24_csoS1-4_cPCR_08_22.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/c/c7/Bielefeld-CeBiTec_2014-10-14_OprF_PCR_08_19.png" height="230px"></a><br><font size="1">Agarose gel from colony PCR. As a Ladder we used <a href="http://www.thermoscientificbio.com/nucleic-acid-electrophoresis/generuler-1-kb-dna-ladder-ready-to-use-250-to-10000-bp">GeneRuler™ 1 kb DNA Ladder from Thermo Scientific</a>. </font> | ||
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<li>Connection of <i>pR6K</i> and <i>oprF</i> | <li>Connection of <i>pR6K</i> and <i>oprF</i> | ||
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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 connected the pR6K-cassette to <i>oprF</i> and amplified and purified both again seperatly | ||
<ul> | <ul> | ||
| - | <li> | + | <li>Connection of pR6K-cassette and <i>oprF</i></li> |
<ul> | <ul> | ||
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> to connect the <i>pR6K</i>-cassette and <i>oprF</i> | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> to connect the <i>pR6K</i>-cassette and <i>oprF</i> | ||
Latest revision as of 22:13, 15 October 2014
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July |
 |
- FumA
- This week we amplified the pSB1C3 backbone of FumA to use it for Gibson Assembly afterwards
- PCR amplification of FumA ( pSB1C3_pre_Fum_A, pSB1C3_suf_Fum_A )
- Annealing temperature: 55 °C
- Bands are as expected (2070 bp)
- FumBCD
- This week we amplified the pSB1C3 backbone of FumBCD to use it for Gibson Assembly afterwards
- PCR amplification ( pSB1C3_pre_Fum_BCD, pSB1C3_suf_Fum_BCD )
- Annealing temperature: 55 °C
- Bands are as expected (2070 bp)
- 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.
- RedET plasmid
- Transformation with electrocompotetent cells of the RedET plasmid into E. coli 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 (BBa_K1172507)
- 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
- Deletion of dcuB and integration of oprF into chromosome
- This week we connected the pR6K-cassette to oprF and amplified and purified both again seperatly
- 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
