Team:Berlin/Project

From 2014.igem.org

(Difference between revisions)
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       <p>     
         <a name="flow-chart">&nbsp;</a>
         <a name="flow-chart">&nbsp;</a>
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               Flowchart, jo!
+
               <img src="https://static.igem.org/mediawiki/2014/e/ee/Team_berlin_flow-chart-01.png" alt="" />
     </div>
     </div>
   </div>     
   </div>     
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                 <h2 class="sub-content-project-headline">Week 1: 02.04.2014 – 06.04.2014</h2>
                 <h2 class="sub-content-project-headline">Week 1: 02.04.2014 – 06.04.2014</h2>
                     Cultivation of E. coli Nissle 1917<br/>
                     Cultivation of E. coli Nissle 1917<br/>
-
                     <br/>
+
                      
                     Genomic DNA extraction of E.coli Nissle strain<br/>
                     Genomic DNA extraction of E.coli Nissle strain<br/>
-
                     <br/>
+
                      
                     Production of BfR, FTNA1 and FTNA2<br/>
                     Production of BfR, FTNA1 and FTNA2<br/>
                     Restriction digest<br/>
                     Restriction digest<br/>
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                 <h2 class="sub-content-project-headline">Week 2: 07.04.2014 – 13.04.2014</h2>
                 <h2 class="sub-content-project-headline">Week 2: 07.04.2014 – 13.04.2014</h2>
                     Colony PCR to check the results<br/>
                     Colony PCR to check the results<br/>
-
                     <br/>
+
                      
                     Bfr, FTNA 1, FTNA 2 primar designed for amplification<br/>
                     Bfr, FTNA 1, FTNA 2 primar designed for amplification<br/>
-
                     <br/>
+
                      
                     Transformation of pQE_80L into DH5α<br/>
                     Transformation of pQE_80L into DH5α<br/>
-
                     <br/>
+
                      
                     Cultivation of BfR, FTNA 1, FTNA 2 in LB<br/>
                     Cultivation of BfR, FTNA 1, FTNA 2 in LB<br/>
               </div>
               </div>
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                 <h2 class="sub-content-project-headline">Week 3: 14.04.2014 – 20.04.2014</h2>
                 <h2 class="sub-content-project-headline">Week 3: 14.04.2014 – 20.04.2014</h2>
                     Miniprep of the cells  from Week 2 <br/>
                     Miniprep of the cells  from Week 2 <br/>
-
                     <br/>
+
                      
                     DNA concentration determination and sequencing<br/>
                     DNA concentration determination and sequencing<br/>
-
                     <br/>
+
                      
                     Expression of BfR, FTNA 1, FTNA 2 and induction with IPTG<br/>
                     Expression of BfR, FTNA 1, FTNA 2 and induction with IPTG<br/>
-
                     <br/>
+
                      
                     Production of a Mutaflor-Supression culture and streaking<br/>
                     Production of a Mutaflor-Supression culture and streaking<br/>
               </div>
               </div>
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                 <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/Primar and Parameter check with Q5 Polymerase and Phusion Polymerase<br/>
                     PCR Plasmid/Primar and Parameter check with Q5 Polymerase and Phusion Polymerase<br/>
-
                     <br/>
+
                      
                     Miniprep of [pKD46+ DH5α] and [pKD4 + DH5 α]<br/>
                     Miniprep of [pKD46+ DH5α] and [pKD4 + DH5 α]<br/>
                     Genomic DNA extraction from Pseudomonas putida<br/>
                     Genomic DNA extraction from Pseudomonas putida<br/>
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                 <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 databse in LB medium and Midiprep<br/>
                     Preculture of strains from Budisa strain databse in LB medium and Midiprep<br/>
-
                     <br/>
+
                      
                     Canamycin cassette PCR of pKD4<br/>
                     Canamycin cassette PCR of pKD4<br/>
-
                    <br/>
+
                                        Restriction digest of Plasmid pKD4 and pKD46v<br/>
-
                    Restriction digest of Plasmid pKD4 and pKD46v<br/>
+
               </div>             
               </div>             
               <div class="sub-content-project">
               <div class="sub-content-project">
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                 <h2 class="sub-content-project-headline">Week 8: 19.05.2014 – 25.05.2014</h2>
                 <h2 class="sub-content-project-headline">Week 8: 19.05.2014 – 25.05.2014</h2>
                     Gel extraction of FieF PCR<br/>
                     Gel extraction of FieF PCR<br/>
-
                     <br/>
+
                      
                     Gene Knockout of FieF in [RV + pKD46] and [WM10+pKD46]<br/>
                     Gene Knockout of FieF in [RV + pKD46] and [WM10+pKD46]<br/>
-
                     <br/>
+
                      
                     Transformation of pKD46 in Nissle and MG 1655<br/>
                     Transformation of pKD46 in Nissle and MG 1655<br/>
-
                     <br/>
+
                      
                     PCR of ATPCS and PPMT<br/>
                     PCR of ATPCS and PPMT<br/>
-
                     <br/>
+
                      
                     Digestion of pQE_80L for cloning<br/>
                     Digestion of pQE_80L for cloning<br/>
               </div>
               </div>
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                 <h2 class="sub-content-project-headline">Week 9: 26.05.2014 – 01.06.2014</h2>
                 <h2 class="sub-content-project-headline">Week 9: 26.05.2014 – 01.06.2014</h2>
                     Production of LB plates<br/>
                     Production of LB plates<br/>
-
                     <br/>
+
                      
                     AMB-1 and Microfluidic Chip were picked up from Max-Plank Institute<br/>
                     AMB-1 and Microfluidic Chip were picked up from Max-Plank Institute<br/>
               </div>
               </div>
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                 <h2 class="sub-content-project-headline">Week 10: 02.06.2014 – 08.06.2014</h2>
                 <h2 class="sub-content-project-headline">Week 10: 02.06.2014 – 08.06.2014</h2>
                     Colony PCR and analytical gel electrophoresis for identifying the right clones<br/>
                     Colony PCR and analytical gel electrophoresis for identifying the right clones<br/>
-
                     <br/>
+
                      
                     PCR of ATPCS and PPMT to identify the correct amplification parameter.<br/>
                     PCR of ATPCS and PPMT to identify the correct amplification parameter.<br/>
-
                     <br/>
+
                      
                     Colony PCR for Knockout.<br/>
                     Colony PCR for Knockout.<br/>
-
                     <br/>
+
                      
                     PCR of ATPCS and PPMT with Q5 High Fidelity Mastermix.<br/>
                     PCR of ATPCS and PPMT with Q5 High Fidelity Mastermix.<br/>
               </div>
               </div>
               <div class="sub-content-project">
               <div class="sub-content-project">
-
                 <h2 class="sub-content-project-headline">week one!</h2>
+
                 <h2 class="sub-content-project-headline">Week 11: 09.06.2014 – 15.06.2014</h2>
-
                  Furthermore, we will work with other metal binding proteins such as metallothioneins and phytochelatin synthases in order to achieve nanoparticle synthesis. Once we have discovered the best way to magnetize E. coli bacteria, we will build and characterize suitable BioBricks that can be used by any research lab or iGEM team in the world in order to remote control the cellular movement.
+
Refine the PCR of PPMT and ATPCS with DMSO<br/>
-
               </div>            
+
              </div>
 +
              <div class="sub-content-project">
 +
                <h2 class="sub-content-project-headline">Week 12: 16.06.2014 – 22.06.2014</h2>
 +
PCR amplification of ATPCS from cDNA<br/>
 +
Colony PCR to seperate cells with FieF Knockout<br/>
 +
Restrcition digest of pQE_80L and ATPCS-PCR Fragment<br/>
 +
Test expression of Ferritin in RV308 (pSB1C3_Ferritin) and Nissle (pSB1C3_Ferritin)<br/>
 +
              </div>
 +
              <div class="sub-content-project">
 +
                <h2 class="sub-content-project-headline">Week 13: 23.06.2014 – 29.06.2014</h2>
 +
Amplification of ATPCS from cDNA and PCR Purification<br/>
 +
Preparation of Nissle, Nissle + Ferritin, RV 308, Rv 308 + Ferritin pre-cultures<br/>
 +
Transformation of human Ferritin on PC514 to Nissle and RV 308<br/>
 +
Induction of Ferritin expression with IPTG<br/>
 +
              </div>
 +
              <div class="sub-content-project">
 +
                <h2 class="sub-content-project-headline">Week 14: 30.06.2014 – 06.07.2014</h2>
 +
Colony PCR and Ligation of ATPCS<br/>
 +
Knockout of FieF and FUR in RV308 and Nissle<br/>
 +
Transformation of  RFP device and Ferritin in RV308, Nissle and WM110 strains.<br/>
 +
PCR amplification of knockout cassette FUR/FieF<br/>
 +
Expression of RV308 (RFP) and RV308 (RFP + Ferritin) after Induction with IPTG<br/>
 +
              </div>
 +
                <div class="sub-content-project">
 +
                <h2 class="sub-content-project-headline">Week 15: 07.07.2014 – 13.07.2014</h2>
 +
Preparation of cryostocks and pre-cultures of ATPCS clone 1-10, WM 110, RV 308, Nissle<br/>
 +
PCR of PPMT with goTaq Polymerase<br/>
 +
Miniprep of ATPCS clone number 3<br/>
 +
              </div>
 +
              <div class="sub-content-project">
 +
                <h2 class="sub-content-project-headline">Week 16: 14.07.2014-20.07.2014</h2>
 +
Midiprep of pQE_80L , PMA-T_PPMT, pKD46  <br/>
 +
Digestion of pQE_80L with Hind III and SacI<br/>
 +
              </div>
 +
              <div class="sub-content-project">
 +
                <h2 class="sub-content-project-headline">Week 17: 21.07.2014-27.07.2014</h2>
 +
Miniprep and Restriction of pBADex-mYFP Venus (Amp); pEx-HisII (Amp); pJS418_Phagemid (dummy) (Cm)<br/>
 +
Ligation of PPMT Fragment from pMAC_PPMT into pEX_HisII<br/>
 +
              </div>
 +
              <div class="sub-content-project">
 +
                <h2 class="sub-content-project-headline">Week 18: 28.07.2014-3.08.2014</h2>
 +
Degradation test of prepped TB-Expression Plasmids <br/>
 +
Transformation of pEX_His_PPMT in MG1655, DH5α, DH10B strains<br/>
 +
Colony PCR of the PPMT clones<br/>
 +
Miniprep and Sequencing of pEX_His_PPMT in DH5 α<br/>
 +
Ligation of ATPCS PCR Fragment into pQE_80 L<br/>
 +
Colony PCR of pQE_80L_ATPCS<br/>
 +
              </div>
 +
              <div class="sub-content-project">
 +
                <h2 class="sub-content-project-headline">Week 19: 04.08.2014 -10.08.2014</h2>
 +
Preculture of ATPCS clones for Sequencing <br/>
 +
PCR of BamHI_PPMT_GS, GS_ATPCS_HindIII, BamHi_HuFerritin_HindIII<br/>
 +
Transformation of  BFR M52H in DH10B.<br/>
 +
              </div>
 +
              <div class="sub-content-project">
 +
                <h2 class="sub-content-project-headline">Week 20: 11.08.2014 - 17.08.2014</h2>
 +
Prepare chemically competent cells<br/>
 +
Generation of Heme-free BFR by Site-directed Mutagenesis<br/>
 +
Digestion of various PCR fragements for cloning into pQE_80L<br/>
 +
Colony PCR of pQE_80L<br/>
 +
Clone Sequencing<br/>
 +
              </div>
 +
              <div class="sub-content-project">
 +
                <h2 class="sub-content-project-headline">Week 21: 18.08.2014 – 24.08.2014</h2>
 +
SDS-PAGE with Coomasie staining for identification of protein expression<br/>
 +
              </div>
 +
              <div class="sub-content-project">
 +
                <h2 class="sub-content-project-headline">Week 22: 25.08.2014 – 31.08.2014</h2>
 +
Calibration curve for iron concentration measurement<br/>
 +
              </div>
 +
              <div class="sub-content-project">
 +
                <h2 class="sub-content-project-headline">Week 23: 15.09.2014 – 21.09.2014</h2>
 +
Preparing cultures for fluorescence microscopy<br/>
 +
Constructing pQE_80L_T5_ATPCS_lac_PPMT<br/>
 +
Digest and Dephosphorylation of vector pQE_80L_ATPCS<br/>
 +
PCR of Biobrick parts (BB0-BB3)<br/>
 +
Digest of pSB1C3-Ferritin<br/>
 +
              </div> 
 +
              <div class="sub-content-project">
 +
                <h2 class="sub-content-project-headline">Week 24: 22.09.2014 – 28.09.2014</h2>
 +
Preparation of pre-cultures<br/>
 +
Checking insertion of gene in ATPCS_PPMT clones by colony PCR<br/>
 +
Digestion of miniprepped pSB1C3_Ferritin (Calgary) for extraction of vector for Biobrick preparation<br/>
 +
Mutagenesis of ATPCS in different plasmid_ATPCS construct<br/>
 +
Berlin Vector – pQE-80L-JBFS- huFerritin Assembly PCR<br/>
 +
Isolation of plasmid DNA of pQE80L_ATPCSMut_GS_PPMT and pQE80L_ATPCS_PPMT <br/>
 +
Repeat PCR of Biobricks<br/>
 +
              </div> 
 +
              <div class="sub-content-project">
 +
                <h2 class="sub-content-project-headline">Week 25: 29.09.2014 – 05.10.2014</h2>
 +
Digestion of Biobrick parts<br/>
 +
Ligation of Biobrick parts with pSB1C3 backbone<br/>
 +
Plasmid Digestion and checked with PCR<br/>
 +
              </div> 
 +
              <div class="sub-content-project">
 +
                <h2 class="sub-content-project-headline">Week 25 06.10.2014 – 12.10.2014</h2>
 +
                  Colony PCR<br/>
 +
                  Miniprep of cultures and preparation of sequencing samples<br/>
 +
                  Preculture of knockouts<br/>
 +
                  PCR of pSB1C3 backbone<br/>
 +
                  Extraction of PCR Product (pSB1C3 linearised)<br/>
 +
                  Iron concentration measurement in knockout strains<br/>
 +
               </div>                                                                                                            
     </div>
     </div>
   </div>     
   </div>     
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       <p>     
       <p>     
         <a name="notebook">&nbsp;</a>
         <a name="notebook">&nbsp;</a>
-
               Notebook, jo!
+
               <div class="sub-content-project">
 +
                <h2 class="sub-content-project-headline">04/02 Wednesday</h2>
 +
== <big><big>Cultivating E. coli Nissle 1917</big></big> ==
 +
 
 +
LB plates were produced without antibiotic. Therefore 10 ml MQ-H<sub>2</sub>O were sterile filtered in 15 ml falcon tube. 1 capsule Mutaflor 331800 was solved in the sterile water.
 +
The E. coli Nissle solution was incubated at 37°C and 200 rpm for 1 h and crossed out at 4 plates to get different dilution of cells.
 +
 
 +
{| class="wikitable"
 +
|-
 +
! dilution !! E. coli Nissle solution !! H<sub>2</sub>O !! dilution factor
 +
|-
 +
| I || 2 µl || 1998 µl || 1:10<sup>3</sup>
 +
|-
 +
| II || 100 µl from dilution I || 900 µl ||1:10<sup>4</sup>
 +
|-
 +
| III || 10 µl from dilution II || 990 µl || 1:10<sup>5</sup>
 +
|}
 +
              </div> 
     </div>
     </div>
   </div>     
   </div>     

Revision as of 21:57, 14 October 2014

1

What is it all about?

  As previous iGEM teams have shown, synthesizing fully functional magnetosomes in E. coli is highly difficult as more than 60 highly regulated genes are involved. As a more feasible alternative, we simply want to synthesize magnetic nanoparticles in E. coli in order to attract cells with strong magnetic fields.
Therefore we want to use different strategies including manipulation of the iron homeostasis of E. coli, expression of different metal binding proteins such as ferritins and metallothioneins as well as a high-throughput growth medium optimization.

Furthermore, we will work with other metal binding proteins such as metallothioneins and phytochelatin synthases in order to achieve nanoparticle synthesis. Once we have discovered the best way to magnetize E. coli bacteria, we will build and characterize suitable BioBricks that can be used by any research lab or iGEM team in the world in order to remote control the cellular movement.

2

Visualisation

  Animation, jo!

3

Detailes Description

  MORE DETAILS, jo!

4

Flow Chart

 

5

The Results

  Results, jo!

6

Lab Summary

 

Week 1: 02.04.2014 – 06.04.2014

Cultivation of E. coli Nissle 1917
Genomic 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 results
Bfr, FTNA 1, FTNA 2 primar 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 2
DNA 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

Minirep

Week 5: 28.04.2014 – 04.05.2014

PCR Plasmid/Primar and Parameter check with Q5 Polymerase and Phusion Polymerase
Miniprep 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 databse in LB medium and Midiprep
Canamycin 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 strains

Week 8: 19.05.2014 – 25.05.2014

Gel extraction of FieF PCR
Gene 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 plates
AMB-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 clones
PCR 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 DMSO

Week 12: 16.06.2014 – 22.06.2014

PCR amplification of ATPCS from cDNA
Colony PCR to seperate cells with FieF Knockout
Restrcition 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 Purification
Preparation 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 ATPCS
Knockout 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, Nissle
PCR 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, pKD46
Digestion 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 Plasmids
Transformation 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 Sequencing
PCR 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 cells
Generation of Heme-free BFR by Site-directed Mutagenesis
Digestion of various PCR fragements for cloning into pQE_80L
Colony PCR of pQE_80L
Clone Sequencing

Week 21: 18.08.2014 – 24.08.2014

SDS-PAGE with Coomasie staining for identification of protein expression

Week 22: 25.08.2014 – 31.08.2014

Calibration curve for iron concentration measurement

Week 23: 15.09.2014 – 21.09.2014

Preparing cultures for fluorescence microscopy
Constructing 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-cultures
Checking 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 parts
Ligation of Biobrick parts with pSB1C3 backbone
Plasmid Digestion and checked with PCR

Week 25 06.10.2014 – 12.10.2014

Colony PCR
Miniprep of cultures and preparation of sequencing samples
Preculture of knockouts
PCR of pSB1C3 backbone
Extraction of PCR Product (pSB1C3 linearised)
Iron concentration measurement in knockout strains
7

Notebook

 

04/02 Wednesday

== Cultivating E. coli Nissle 1917 == LB plates were produced without antibiotic. Therefore 10 ml MQ-H2O were sterile filtered in 15 ml falcon tube. 1 capsule Mutaflor 331800 was solved in the sterile water. The E. coli Nissle solution was incubated at 37°C and 200 rpm for 1 h and crossed out at 4 plates to get different dilution of cells. {| class="wikitable" |- ! dilution !! E. coli Nissle solution !! H2O !! dilution factor |- | I || 2 µl || 1998 µl || 1:103 |- | II || 100 µl from dilution I || 900 µl ||1:104 |- | III || 10 µl from dilution II || 990 µl || 1:105 |}
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