06/19 Thursday

             PCR-program: 

             Elongation time: 2Min, Annealing Temperature: 56°C

              Magnetization of E. coli by expression of human Ferritin, 1st Experiment part 1 of 4 

             Aim: 
             Test the possibility of Magnetization of E.coli cells by the expression of human Ferritin:

             Procedure:

             To evaluate the effect of Ferritin  on the Magnetization different cultures were compared:

             For Both RV308 and Nissle

             - Transform the Human Ferritin gen carried on the Plasmid PC514 provided from (iGEM Team of Calgary university, Canada) to E. coli strains
             (Nissle and  RV308) via electroporation, plate on (Amp) plates for selection.
 
              

06/20 Friday Restriction digest of pQE_80L and ATPCS-PCR fragment

             5x Mastermix = Mastermix for ATPCS 

             Enzymes were added to each single aliquot and not into the Mastermix

             Incubation: for 1,5 h at 37 °C

             Deactivation: 80°C for 5 min

             I used the wrong restriction enzymes as I got confused with the PCR fragment. Digestion with SacI and HindIII is needed not  BamHI.
             Repeat PCR for ATPCS as well as restriction digest!

             Test expression of Ferritin in RV308 (pSB1C3_Ferrtin) and Nissle (pSB1C3_Ferrtin)

             5ml overnight culture used as an inoculum.

             4 ml culture were diluted to 40 ml with LB and grown for 1 h at 37°C until an OD600 of 0.7 and 0.8 was reached.

             Non-induced SDS sample was taken and induced with 1 mM IPTG

             After expression for 3 h induced SDS sample was taken.
             SDS PAGE
             Ferritin band expected at 21kDa


             Key:

             - 1st band: Nissle (non induced)

             - 2nd band: RV308 (non induced)

             - 3rd band: Ladder

             - 4th band: Nissle (induced)

             - 5th band: RV308 (induced)

06/25 Wednesday - Amplification of ATPCS (cDNA)

             PCR-program

             PCR Purification
             -Purification Kit 

             →DNA concentration measurement showed, that the Absorbance ratio of 260nm:280nm is quite low, which means that targeted DNA got contaminated.

             Preparation of pre-culture
             Strains:

             - e.coli nissle

             - e.coli nissle + ferritin

             - RV308

             - RV308 + ferritin

             Assay
             - 5ml LB media + 1 picked clone of the pre-day

             - Addition of 5µl CN37 to the strains with ferritin

             Incubation: 37°C; 170rpm; o/n
              
              

06/27 Friday

06/28 Saturday

             - Transfer the Precultures (OD= 0.4-0.5) to 25, 50 mL flasks (half filled with LB) and cultivate them at 37°C for 3 hours
             RV308 (+,+,+) & (-,+,+) And Nissle (+,+,+) were cultivated in 50 mL flasks

              Strain:  OD

              RV308    0,78

              Nissle   0,86

             - Induction with (20µl (50 ML flasks)/10µl (25 ML flasks)) IPTG (concentration 1 M).

             - Add FeCl2 and MnCl2 (100µL in 50ml flasks/ 50µl in 25ml flasks) Incubation over night at 37C°

              Strain:  OD

              RV308    2.56

              Nissle   2.8

               
              

06/29 Sunday

             Evaluation:

             SDS Page:  was not clear! 

             Magnetization :
             

             RV308                                             

             PCR program

             Gel Electrophoresis
             1% Agarose gel with EtBr; 8µl sample; 5µl Ladder

             "Key:"

             First band= Ladder

              
              Each other band is a different picked clone (10 clones were picked)

              

              

07/01 Tuesday - ATPCS Ligation

07/02 Wednesday - Knockout of Fief and Fur in RV308 and Nissle

             Transformation of stains RV, Nissle, WM

             The stains RV308[ferritin], Nissle[ferritin], RV308, Nissle and WM110 were transformed with RFP. RV308[ferritin] and Nissle[ferritin] were deleted on CM+Amp plates and 5 ml culture was added with CM/Amp and incubated at 37°C. The transformed strains RV308, Nissle and WM110 were deleted on Amp plates and 5 ml culture was added with Amp and incubated at 37°C.

             Results of transformation

The cultures of WM110 1 + RFP and WM110 2 + RFP were deleted on Amp plates and incubated at 37°C. Moreover 5 ml precultures from RV308 + RFP + Amp and RV308[ferritin] + RFP + CM+Amp were prepared.

07/03 Thursday - PCR amplification of knockout cassette FUR/FieF

             The amplification was performed with the program in the table below.

             --> hold at 8°C

             The amplification results were analyzed in agarose gelelectrophoresis (1% agarose; 0,5µl Gel Red; 10x diluted ladder; 6x loading dye).

07/04 Friday - Expression von RV308 (RFP) und RV308 (RFP + Ferritin)

             *Fe/Mn were solubilized by autoclaving and 1M stock solution

Although not planned IPTG and metal ion solutions were added simultaneously due to time constraints. It was planned to add metal ions 2h after induction

07/07 Monday - Preculture prepared of:

07/09 Wednesday - PCR for PPMT with goTaq (Promega)

07/17 Thursday - Midiprep

07/24 Thursday - miniPrep + restriction PPMT und iGEM plasmids

07/25 Friday

Ligase was deactivated and ligation transformed after weekend

07/28 Monday

07/29 Tuesday

             Ligase was deactivated and ligation transformed next day

             <img src="" alt="" />

             #pMAC_PPMT*
             #pQE_80L*
             #pSB1c3
             #pKD46*

(*)Bands at 20 kbp for Promega prepped Plasmids.

07/30 Wednesday

              Transformation 
             Chemical competent cells XL Blue 
             * 3 µl for each Ligation Assay
             * 10 min incubation on ice
             *60 s Heat schock
             * 2 min incubation on ice
             *950µl LB added
             *1h --> 37 °C for Recovery
             *platted on Amp-plates
              Eporation 
             *0.2 µl Ligation assay in competent DH5α [iGEM]
             *1.7 kV
             * 1h; 37°C
             * platted on Amp plates
              
              

07/31 Thursday

08/06 Wednesday

             Program:

Analytical Gel Expected Bands clearly seen for PPMT and Ferritin. Only little ATPCS band shows Primer Dimer inhibit PCR. Anyhow we will try a Gel extraction and after a Assembly PCR Generation of Heme-free BFR by Site-directed Mutagenesis, part 1 of 5
Site-directed mutagenesis was performed by the QuickChange method
Primer design with http://www.bioinformatics.org/primerx/ methionine (on position 52) was substituted by histidine BFR M52H

08/07 Thursday

08/08 Friday - Gradienten PCR GS_ATPCS_HindIII

             PCR

             Program:

             - preparative agarose gel 1% and gel ex of GS_ATPCS_HindIII bands at ca. 1490 bp

             - accidentally also loaded BamHI_PPMT_GS onto Gel and was also  extracted (elution with 25 µl elution buffer).

             - BamHI_Ferritin_HindIII was PCR purified

             - DNA concentration measurement for  purified pcr fragments:

             GS_ATPCS_HindIII = 33 ng/µl

             BamHI_PPMT_GS = 48 ng/µl

             BamHI_Ferritin_HindII = 125 ng/µl

             Assembly PCR

             An Assembly PCR was used to align BamHI_PPMT_GS with GS_ATPCS_HindIII and amplify the complementary structure in order to form a fusion protein coding sequence.

             Therefore, both fragments were added a templated equal molar amounts.

             Calcultation:

             bpATPCS = 1493

             bpPPMT = 255

             mATPCS/bpATPCS = mPPMT/bpPPMT

             mATPCS = (1ng * 1493) / 255

             mATPCS= 58 ng

             Program:

              Transformation into DH10B 
             #BFR M52H
             # BFR M52H
             *Recover in LB--> 1h at 37°C
             
              

08/11 Monday - Plan-prepare fragments for digest

             *In pQE_80L (Amp)    BamHI_FTH_FTL_Hind III   (PCR Construct available)
             Construct Size 1077 of 4731

             *In pQE_80L (Amp)   Sacl_ATPCS_Hind III    (PCR Construct not available)
             Construct Size 1400 of 4731

             *In pJS418 (CM)    XbaI_PPMT_PstI       (PCR Construct available)
             Construct Size 255 of 3800

             PCR Fragmente PPMT_GS_ATPCS+ATPCS+Purification (Gelex)

After Gelex:
PPMT 48ng/μl
Ferritin 125ng/μl (Pur from PCR)
ATPCS 33ng/μl

08/12 Tuesday - Preparing chemically competent cells

             Digestion

             * 35 ng/L  PPMT ATPCS 1
             * 15ng/L  PPMT ATPCS 2
             * 25ng/L  ATPCS
             * 13ng/L  P_Mac_PPMT
             * 31ng/L  PJS_418
              




 

             Ampicillin  

             JBH1 in VBH

             JBH2 in VBH 

             JSH in VSH 

             Chloramphenicol 

             PPMT in PJS_418

             Transformation in DH10b (cc.c)

             106 Colony for every approach  

              
              

08/13 Wednesday - pQE 80L Colony PCR

             Samples : 

             # PPMT GS ATPCS   size 1912
             # PPMT GS ATPCS   size 1912
             # ATPCS in pQE80L   size 1770
             # Ferritin in pQE80L   size 1376
             # PPMT in PJS   size 420

             pQE80L=300bp  
     
             pJS = 170bp

             Sequence clones A - H + J

              Generation of Heme-free BFR by Site-directed Mutagenesis, part 2 of 5 

             QuickChange Site-Directed Mutagenesis

             20µL approach:

             Template BFR: 

             0,5 µL              A4 (66ng/µL)

             0,2 µL    D2 (144ng/µL)

             PCR Program

             Σ20µL -8µL fürs Gel= 12µL +1µL DpnI 

             37°C 90min

             chemical Transformation (Fabian)
             

             in DH10b

plating out on Amp plates

08/15 Friday - Clone Sequencing

08/19 Tuesday

08/20 Wednesday

08/21 Thursday

08/22 Friday - SDS-PAGE with Coomassie staining

             # Marker
             # Pellet ATPCS/PPMT
             #15:00 after induction ATPCS/PPMT
             #overnight ATPCS/PPMT
             #GS Pellet
             #PGSA after induction 15:00
             #GS P/A overnight
             #human Ferritin Pellet
             #human Ferritin after induction 15:00
             #human Ferritin overnight

             Analysis of Expression

             Human Ferritin = 42 kDa (Potparam  Expasy)

             ATPCS = 56.3 kDA (Potparam  Expasy)

             PPMT = 7.9 kDA (Potparam  Expasy)

             ATPCSGSPPMT = 64.3 kDa (Potparam Expasy)
              
              

08/29 Friday - Calibration curve for Iron concentration measurement

09/03 Wednesday

             Procedure: ...

-Precultures of Nissle and RV308 (wild types)

09/04 Thursday

09/05 Friday

09/06 Saturday

09/11 Thursday

09/12 Friday

09/13 Saturday

09/15 Monday

             -Test the Magnetization using a permanent magnet under Fluorescent microscope. 

             Evaluation 

             -Cells grew
             -no red fluorescence observed
             -no Magnetization observed

             Possible Explanation:
             - Adding Mn ions instead of Fe could be effected the Magnetization 
             - The red Fluorescence gen contains a cadaverin chain, this could explain the non-fluorescence.
              
              

09/16 Tuesday

              Program 

09/17 Wednesday - Digest and Dephosphorylation of vector pQE_80L_ATPCS

Gelextraction resulted in HindIII cut pQE80L_ATPCS 20 µl with 12 ng/µl
In parallel the PCR fragment HindIII_lac_PPMT_HindIII was purified and is ready for digest.

09/18 Thursday

             'Aim
             

             -Double Trafo of the Human-Ferritin and fluorescence marker.

             Procedure:…

             Stains: RV308, Nissle and DH0B (control)

             -chemical Double-biotransformation of:

              -0.5 µl of Human-ferrtin on PQE-80L(490 ng/µl) with (Amp)

              -3 µl green fluorescence protein Nerm ASGP-R-GFP (83 ng/µl) with (Kan)

             for control:

             - 3 µl green fluorescence protein Nerm ASGP-R-GFP (83 ng/µl) with (Kan) is transformed into DH05

             - Double Trafo of (pJS418_PPMT and pQE_80L_ATPCS)

              
              

09/19 Friday

             Primers

             PCR Programms
             BB0 & BB1

             BB2

              



              Biobricks derived from pQE80L constructs

              BB1  BFR  191µg/ml

              BB2  BFRM  173µg/ml 

              BB2  HuFerritin  166 µg/ml

               Magnetization of E. coli by expression of human Ferritin, Bio-transformations, 3rd Experiment part 2of 7 
              -No positive colones were detected for double-trafo expect these for control (transformation was successful in DH05).
              

09/20 Saturday - Digest of PSB1C3-Ferritin

             37°C for 1,5h
             (inactivate for 20min at 80°C)

             Gel electrophoresis 
             1% Agarose-Gel 

             + 3 µL EthBr

             200µL Probe + 40 µL loading dye (6x)

             5 µL GenRuler-Mix

             no Gel-extraction, because nothing was seen on the gel, except the marker (GenRuler) 

              Magnetization of E. coli by expression of human Ferritin, Bio-transformations, 3rd Experiment part 3 of 7 
             -Transformation of RF (red fluorescence protein) on with (Kan) into DH05
              
              

09/21 Sunday

09/22 Monday - Precultures

Dissolved colonies 4 and 6 which showed band at 400 bp were grown each in a cultivation tube with LB+Amp100 at 37°C

09/23 Tuesday - Mutagenesis of ATPCS

             ... Details Saba....

             After digest with DpnI the reaction was transformed into competent DH10b E. coli cells via heat shock transformation.

             After streaking out all aliquots on LB-Amp Agar plates, they were incubated at 37 °C over night.

              Digest of miniprepped PSB1C3_Ferritin (Calgary) for extraction of vector for BioBrick preparation 

             

             The reaction was incubated at 37 °C for 1,5 h.

             The digested vector was purified using a 1% agarose gel and the Roth GelNebulizer purification kit. The vector band was expected at 2000 bp and the insert band at about 1000 bp.

             both colonies from Saba were grown --> Mini-prep (Johann) to harvest plasmid DNA

             Mutagenesis of ATPCS
             in 
                pQE80L_ATPCS-GS-PPMT

                pQE80L_ATPCS_PPMT

                pQE80L_ATPCS

             3x PCR-Reactions

                Q5 MM   12.0µl

                PB_for SN_ATPCS_xbaI_Mut_for   1.25µl

                PB_rev SN_ATPCS_xbaI_Mut_rev   1.25µl

                nuc.-free H2O   10.5µl

                template (1ng)   ~ 1.0µl (dilations of original plasmids)

             Total 26µl

             PCR Program

             Trafo of 5µl PCR-Product in DH10b cells --> incubation at 37 °C

              Magnetization of E. coli by expression of human Ferritin, Bio-transformations, 3rd Experiment part 5 of 7 

             - Preculture of DH05 with RF plasmid

09/24 Wednesday

             * 1% Agarose, Ethidium bromide 

             * 50µL Assembly PCR product + 10µL 6*DNA dye
             * 10µL GeneRuler DNA Ladder Mix (ThermoScientific)
             * Gel Electrophoresis conditions 100V, 30min
              


             


              Gel Extraction 

              Gel Extraction using GeneJET Gel Extraction Kit (ThermoScientific) 
 
             * Determination of Concentration by Ultraviolet Spectrophotometry

             ?

             Double Digest 

             * Digestion of 1μg of purified Insert DNA using FastDigest BamHI / HindIII
             * 1h at 37°C
             ?

             * Heat inactivation of FastDigest BamHI / HindIII, 15min at 80°C

             PCR purification 

             * PCR purification of BamHI / HindIII digested insert using GeneJET PCR Purification Kit (ThermoScientific)
             * Determination of Concentration by Ultraviolet Spectrophotometry

             ?

             Ligation 

             * Using the Molar Ligation Ratio 1:5 (1 times vector and 5 times insert)

             * 20min at RT

             Chemical Transformation 

             * 100µL Aliquots of Chemically Competent Cells - DH10B E.coli + 10µL Ligation approach chilled on ice for 30 min
             * Heat shock for 1min at 42°C
             * Add 900µL preheated (37°C) LB
             * Incubate the cells for 1h at 37°C with shaking approx. 200rpm
             * Plate 200µL and spin down the rest and plate on LB Ampicillin, incubate overnight at 37°C

             Saba
             o/n culture of clones from successful pQE80L_ATPCS-mut_GS_PPMT and pQE80L_ATPCS-mut_PPMT transformation
             --> 37°C, 200pm, o/n 5-6 nl LB+AB (Amp)

              Magnetization of E. coli by expression of human Ferritin, Bio-transformations, 3rd Experiment part 6 of 7 

             - Miniprep of the Preculture, DNA concentration 71 ng/µl

             Chemical Biotransformation of:
              -3 µl of RFP 71 ng/µl
              -2 µl Ferritin jbfs_m (different ferrtin)(120 ng/µl) with  (Amp)'

             in RV308 and Nissle (2 plates each)
              
              

09/25 Thursday - PCR of Biobrick Parts

             PCR-Ansätze V/µl
                Q5 2xMM       25.0
                10µM PB_fw    2.5
                10µM PB_rew   2.5
                template      2.0 (~1ng)
                nuc.-free H2O 18.0
             PCR-Programm

             PCR purification of 1,2, 3 (Jbfs_mil_Ferritin_cp),BF-cp and BFM-cp biobrick parts

Magnetization of E. coli by expression of human Ferritin, Bio-transformations, 3rd Experiment part 7 of 7
- Double-trafo was successful in all RV308 plates but not in Nissle

09/30 Tuesday

10/01 Wednesday - Plasmid Digestion & PCR Probe

PCR Probe Ligation plates evaluated:
All plates had clones except for BFR_cp from each plate transformation one clone was cultivated with LB/Cm (~5ml) o/n at 37°C

10/02 Thursday - Cultures were Mini-preped

10/06 Monday- Colony PCR

             Colony-PCR of other clones

             PCR-Programm

             --> gel (1% Agarose)

             Clones 3, 5, 6, 7 and 13 were grown o/n in LB/Cm for Mini-prep on the following day. therefore 5µl of the dissolved clones were used for each inoculation

             --> 37°C, 200rpm

2x M9 Media Production
(Christina)
2X M9 mineral medium
For 500 ml M9 mineral medium add to XXX ml sterile water:

             KH2PO4

             NaCl

             NH4Cl || 67.4mM

             44.0mM

             17.10mM

             18.70mM

10/07 Tuesday

10/08 Wednesday - PCR of PSB1C3 Backbone

              Extraction of PCR Product (PSB1C3 linearized) 

             After extracting the expected band at 2000 bp it was purified using the EMDMillipore Montage Gel Extraction Device.

             Resulting in about 120 µl DNA extract in TAE buffer with an concentration of 9ng/µl

             * 1.5 hours at 37°C
             * PCR Purification
             * DNA concentration measurement = 35ng/µl

              Ligation 
             Calculation
             *BFM52H = 3.22µl
             *BF_cp= 6.7µl
             *jbfs_m = 8.06 µl
             *BB_AnP = 10.64 µl
             * HuFer = 9.45 µl
              Iron Concentration measurement 
             278 mg FeSO4.7H2O dissolved in 10 ml d.H2O 

             λ FieF PCP2 Amp  1:100 ---> 3.0 OD

             iGEM BV125 tet 1:100 --> 3.0 OD 

             λ FieF PCP1 Amp 1:20 -->3.26 OD
             iGEM BV125 tet 1:20---> 2.44

             too high OD!

             Both samples diluted 1:2
             Samples
             #  FieF + 5µl 0.1 M Fe-stock solution
             # FieF + 15 µl 0.1 M Fe-Stock solution
             # BV + 5 µl 0.1M Fe-stock solution
             # BV + 15 µl 0.1 M Fe-stock solution

             Overnight incubation at 30°C on a shaker 
     
              

10/09 Thursday

             For Sequencing:
             *total volume: 15µl
             *Primer used: PB795 3µl each Sequencing approach
             *Sample [µl] see table
             *nucl.free Water [µl] see table

             evaluation of Trafos ---> no growth --->Repeat Trafo in other cells
 
              

10/10 Friday - STRATEGY 1 - Knockout of FieF and FUR Genes

             --> Annealing Temperature: 57°C

             FUR

             --> Annealing Temperature: 57°C
              PCR-Assay 

              PCR-Program 

             
               Gel-Electrophoresis 

              1% Agarose-Gel + 1µl GelRed; 90V; 30min


              

10/14 Tuesday - Iron-Assay