"
Team:Berlin/Project/Summary
From 2014.igem.org
(Difference between revisions)
m |
|||
| Line 106: | Line 106: | ||
Colony PCR to check the results<br/> | Colony PCR to check the results<br/> | ||
| - | Bfr, FTNA 1, FTNA 2 | + | Bfr, FTNA 1, FTNA 2 primer designed for amplification<br/> |
Transformation of pQE_80L into DH5α<br/> | Transformation of pQE_80L into DH5α<br/> | ||
| Line 124: | Line 124: | ||
<div class="sub-content-project"> | <div class="sub-content-project"> | ||
<h2 class="sub-content-project-headline">Week 4: 21.04.2014 – 27. 04.2014 </h2> | <h2 class="sub-content-project-headline">Week 4: 21.04.2014 – 27. 04.2014 </h2> | ||
| - | + | Miniprep | |
</div> | </div> | ||
<div class="sub-content-project"> | <div class="sub-content-project"> | ||
<h2 class="sub-content-project-headline">Week 5: 28.04.2014 – 04.05.2014</h2> | <h2 class="sub-content-project-headline">Week 5: 28.04.2014 – 04.05.2014</h2> | ||
| - | PCR Plasmid/ | + | PCR Plasmid/Primer and Parameter check with Q5 Polymerase and Phusion Polymerase<br/> |
Miniprep of [pKD46+ DH5α] and [pKD4 + DH5 α]<br/> | Miniprep of [pKD46+ DH5α] and [pKD4 + DH5 α]<br/> | ||
| Line 136: | Line 136: | ||
<div class="sub-content-project"> | <div class="sub-content-project"> | ||
<h2 class="sub-content-project-headline">Week 6: 05.05.2014 – 11.05.2014</h2> | <h2 class="sub-content-project-headline">Week 6: 05.05.2014 – 11.05.2014</h2> | ||
| - | Preculture of strains from Budisa strain | + | Preculture of strains from Budisa strain database in LB medium and Midiprep<br/> |
Canamycin cassette PCR of pKD4<br/> | Canamycin cassette PCR of pKD4<br/> | ||
| Line 180: | Line 180: | ||
<h2 class="sub-content-project-headline">Week 12: 16.06.2014 – 22.06.2014</h2> | <h2 class="sub-content-project-headline">Week 12: 16.06.2014 – 22.06.2014</h2> | ||
PCR amplification of ATPCS from cDNA<br/> | PCR amplification of ATPCS from cDNA<br/> | ||
| - | Colony PCR to | + | Colony PCR to separate cells with FieF Knockout<br/> |
| - | + | Restriction digest of pQE_80L and ATPCS-PCR Fragment<br/> | |
Test expression of Ferritin in RV308 (pSB1C3_Ferritin) and Nissle (pSB1C3_Ferritin)<br/> | Test expression of Ferritin in RV308 (pSB1C3_Ferritin) and Nissle (pSB1C3_Ferritin)<br/> | ||
</div> | </div> | ||
| Line 234: | Line 234: | ||
Prepare chemically competent cells<br/> | Prepare chemically competent cells<br/> | ||
Generation of Heme-free BFR by Site-directed Mutagenesis<br/> | Generation of Heme-free BFR by Site-directed Mutagenesis<br/> | ||
| - | Digestion of various PCR | + | Digestion of various PCR fragments for cloning into pQE_80L<br/> |
Colony PCR of pQE_80L<br/> | Colony PCR of pQE_80L<br/> | ||
Clone Sequencing<br/> | Clone Sequencing<br/> | ||
| Line 240: | Line 240: | ||
<div class="sub-content-project"> | <div class="sub-content-project"> | ||
<h2 class="sub-content-project-headline">Week 21: 18.08.2014 – 24.08.2014</h2> | <h2 class="sub-content-project-headline">Week 21: 18.08.2014 – 24.08.2014</h2> | ||
| - | SDS-PAGE with | + | SDS-PAGE with Coomassie staining for identification of protein expression<br/> |
</div> | </div> | ||
<div class="sub-content-project"> | <div class="sub-content-project"> | ||
| Line 276: | Line 276: | ||
Preculture of knockouts<br/> | Preculture of knockouts<br/> | ||
PCR of pSB1C3 backbone<br/> | PCR of pSB1C3 backbone<br/> | ||
| - | Extraction of PCR Product (pSB1C3 | + | Extraction of PCR Product (pSB1C3 linearized)<br/> |
Iron concentration measurement in knockout strains<br/> | Iron concentration measurement in knockout strains<br/> | ||
</div> | </div> | ||
Latest revision as of 02:06, 18 October 2014
Explore our Project:
Explore our Project:5
Lab Summary
Week 1: 02.04.2014 – 06.04.2014
Cultivation of E. coli Nissle 1917Genomic DNA extraction of E.coli Nissle strain
Production of BfR, FTNA1 and FTNA2
Restriction digest
PCR Purification
Ligation Assay
Transformation into DH5α-Cells
Week 2: 07.04.2014 – 13.04.2014
Colony PCR to check the resultsBfr, FTNA 1, FTNA 2 primer designed for amplification
Transformation of pQE_80L into DH5α
Cultivation of BfR, FTNA 1, FTNA 2 in LB
Week 3: 14.04.2014 – 20.04.2014
Miniprep of the cells from Week 2DNA concentration determination and sequencing
Expression of BfR, FTNA 1, FTNA 2 and induction with IPTG
Production of a Mutaflor-Supression culture and streaking
Week 4: 21.04.2014 – 27. 04.2014
MiniprepWeek 5: 28.04.2014 – 04.05.2014
PCR Plasmid/Primer and Parameter check with Q5 Polymerase and Phusion PolymeraseMiniprep of [pKD46+ DH5α] and [pKD4 + DH5 α]
Genomic DNA extraction from Pseudomonas putida
Enzyme digestion of different plasmids
Week 6: 05.05.2014 – 11.05.2014
Preculture of strains from Budisa strain database in LB medium and MidiprepCanamycin cassette PCR of pKD4
Restriction digest of Plasmid pKD4 and pKD46v
Week 7: 12.05.2014 – 18.05.2014
Biotransformation of Ferritin and pKD46 in Nissle and DH5 α strainsWeek 8: 19.05.2014 – 25.05.2014
Gel extraction of FieF PCRGene Knockout of FieF in [RV + pKD46] and [WM10+pKD46]
Transformation of pKD46 in Nissle and MG 1655
PCR of ATPCS and PPMT
Digestion of pQE_80L for cloning
Week 9: 26.05.2014 – 01.06.2014
Production of LB platesAMB-1 and Microfluidic Chip were picked up from Max-Plank Institute
Week 10: 02.06.2014 – 08.06.2014
Colony PCR and analytical gel electrophoresis for identifying the right clonesPCR of ATPCS and PPMT to identify the correct amplification parameter.
Colony PCR for Knockout.
PCR of ATPCS and PPMT with Q5 High Fidelity Mastermix.
Week 11: 09.06.2014 – 15.06.2014
Refine the PCR of PPMT and ATPCS with DMSOWeek 12: 16.06.2014 – 22.06.2014
PCR amplification of ATPCS from cDNAColony PCR to separate cells with FieF Knockout
Restriction digest of pQE_80L and ATPCS-PCR Fragment
Test expression of Ferritin in RV308 (pSB1C3_Ferritin) and Nissle (pSB1C3_Ferritin)
Week 13: 23.06.2014 – 29.06.2014
Amplification of ATPCS from cDNA and PCR PurificationPreparation of Nissle, Nissle + Ferritin, RV 308, Rv 308 + Ferritin pre-cultures
Transformation of human Ferritin on PC514 to Nissle and RV 308
Induction of Ferritin expression with IPTG
Week 14: 30.06.2014 – 06.07.2014
Colony PCR and Ligation of ATPCSKnockout of FieF and FUR in RV308 and Nissle
Transformation of RFP device and Ferritin in RV308, Nissle and WM110 strains.
PCR amplification of knockout cassette FUR/FieF
Expression of RV308 (RFP) and RV308 (RFP + Ferritin) after Induction with IPTG
Week 15: 07.07.2014 – 13.07.2014
Preparation of cryostocks and pre-cultures of ATPCS clone 1-10, WM 110, RV 308, NisslePCR of PPMT with goTaq Polymerase
Miniprep of ATPCS clone number 3
Week 16: 14.07.2014-20.07.2014
Midiprep of pQE_80L , PMA-T_PPMT, pKD46Digestion of pQE_80L with Hind III and SacI
Week 17: 21.07.2014-27.07.2014
Miniprep and Restriction of pBADex-mYFP Venus (Amp); pEx-HisII (Amp); pJS418_Phagemid (dummy) (Cm)Ligation of PPMT Fragment from pMAC_PPMT into pEX_HisII
Week 18: 28.07.2014-3.08.2014
Degradation test of prepped TB-Expression PlasmidsTransformation of pEX_His_PPMT in MG1655, DH5α, DH10B strains
Colony PCR of the PPMT clones
Miniprep and Sequencing of pEX_His_PPMT in DH5 α
Ligation of ATPCS PCR Fragment into pQE_80 L
Colony PCR of pQE_80L_ATPCS
Week 19: 04.08.2014 -10.08.2014
Preculture of ATPCS clones for SequencingPCR of BamHI_PPMT_GS, GS_ATPCS_HindIII, BamHi_HuFerritin_HindIII
Transformation of BFR M52H in DH10B.
Week 20: 11.08.2014 - 17.08.2014
Prepare chemically competent cellsGeneration of Heme-free BFR by Site-directed Mutagenesis
Digestion of various PCR fragments for cloning into pQE_80L
Colony PCR of pQE_80L
Clone Sequencing
Week 21: 18.08.2014 – 24.08.2014
SDS-PAGE with Coomassie staining for identification of protein expressionWeek 22: 25.08.2014 – 31.08.2014
Calibration curve for iron concentration measurementWeek 23: 15.09.2014 – 21.09.2014
Preparing cultures for fluorescence microscopyConstructing pQE_80L_T5_ATPCS_lac_PPMT
Digest and Dephosphorylation of vector pQE_80L_ATPCS
PCR of Biobrick parts (BB0-BB3)
Digest of pSB1C3-Ferritin
Week 24: 22.09.2014 – 28.09.2014
Preparation of pre-culturesChecking insertion of gene in ATPCS_PPMT clones by colony PCR
Digestion of miniprepped pSB1C3_Ferritin (Calgary) for extraction of vector for Biobrick preparation
Mutagenesis of ATPCS in different plasmid_ATPCS construct
Berlin Vector – pQE-80L-JBFS- huFerritin Assembly PCR
Isolation of plasmid DNA of pQE80L_ATPCSMut_GS_PPMT and pQE80L_ATPCS_PPMT
Repeat PCR of Biobricks
Week 25: 29.09.2014 – 05.10.2014
Digestion of Biobrick partsLigation of Biobrick parts with pSB1C3 backbone
Plasmid Digestion and checked with PCR
Week 25 06.10.2014 – 12.10.2014
Colony PCRMiniprep of cultures and preparation of sequencing samples
Preculture of knockouts
PCR of pSB1C3 backbone
Extraction of PCR Product (pSB1C3 linearized)
Iron concentration measurement in knockout strains
Sponsors
