Team:Marburg:Project:Notebook:August

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Notebook: August

01.08.2014

18.50 Design of a Strep-Tag/ StrepDARPidin system

Aim: Designing a new system for using the Flagellin-DARPin-Konstrukt

A new plan was made to obtain the flagellas multi-functionality, using an affinity tag. We want to integrate a Strep-Tag into the flagellin so that the DARPin domain can be fused to the flagellum via streptavidin.

The D2 domain of Salmonella typhimurium's flagellin was used as a linker between the hag of Bacillus subtilis and the Strep-Tag, furthermore GSGS linkers were used between the D2 and the StrepTag.

Additionally the DARPin was designed with an N-terminal His-Tag and C-Terminal streptavidin, so a chimeric protein would be generated.

The designed structures were synthesized by Integrated DNA Technologies.

15.61 Design of Hag-D2-Cup

Aim: Designing a new domain construct for expression of cup1-1-Hag

We decided to design the flagellin as well with the D2 domain and the cup1-1.

The designed structures were synthesized by Integrated DNA Technologies

04.08.2014

14.68 Crystallization of Arc1p-C

Aim: Crystallize the Arc1p-C-single domain

For the experiments the sitting drop method in SWISSCI MRC 2 well crystallization plates with a drop size of 1 µL was used (1:1 mixture of protein and crystallization solution) with an reservoir volume of 50 µL. Crystals of Arc1p-C-single were obtained by room temperature and at 6 mg/mL protein concentration after 1 week in 0.1 M MES, pH 6.0, 10% (v/v) glycerol, 30% (w/v) PEG 600 and 5% (w/v) PEG 1000. To collect data the crystals were flash frozen in liquid nitrogen and measured at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France at beamline ID23-1. The structure of Arc1p-C-single was determined by molecular replacement (MR) using the crystal structure of human Arc1p-C (PDB ID: 1FL0).

05.08.2014

18.51 Isolation of flagella from Bacillus subtilis WT3610

Aim: isolation of filaments of WT3610 as negative control for e.g. FACS analysis & microscopy

1 L LB was inoculated with WT 3610 and incubated over night at 37°C.

15.62 Restriction digest of piGEM-005 SpeI

Aim: linearizing vector for Gibson assembly with Hag-D2-Cup and -D2-Strep

Component Volume (µl)
piGEM-005 (100 ng/µL 20
CutSmart 10x 2,5
SpeI 0,2
Water 2,3
Total Volume 25

Incubation at 37°C for 2 hours – Inactivation by heat shock at 85° for 10 min.

End concentration: 50 ng/µL

18.51 NcoI/BamHI & NcoI/XhoI digest of pET24d

Aim: purification of vector for ligation with StrepDARPidin gene

Component Volume [µl] Volume [µl]
pET24d (80 ng/µL) 30 30
CutSmart 10x 3,5 3,5
NcoI 0,25 0,25
BamHI 0,25 -
XhoI - 0,25
Water 1 1
Total Volume 35 35

Incubation at 37°C for 2h .

The fragments were purified via gel extraction.

End concentration: 25 ng/µL pET24d-N/B & 15 ng/µL-N/X

06.08.2014

18.52 Isolation of flagella from Bacillus subtilis WT3610

Aim: isolation of filaments of WT3610 as negative control for e.g. FACS analysis & microscopy

The preculture was centrifuged at 4000 rpm for 15 minutes and the pellet was resuspended in 33 mL TRIS/HCl buffer (pH 8.0) with 0,5% Brij-58. A stock solution of lysozyme with 10 mg/mL was made. 330 µL were added to the TRIS/HCl buffer mix. The lysis was performed at 4°C while rotating the tubes until the pellet was completely solved which took approx. 2 - 3 hours. DNase (5 µg/ mL) in presence of 0,01 M magnesium chloride was added and incubated for half an hour at 4°C in the rotator as well.

After that the suspension was centrifuged at 10000x g for 10 min. 80 µL of the supernatant were transferred into a 1,5 mL tube. The supernatant was then centrifuged at 87000xg for 90min.

The pellet was resuspended in 10 mL standard saline citrate (0,01M trisodium citrate & 0,1M sodium chloride, pH 7,3) on ice and 80 µL were taken as well. For fractioning, 2 g of ammonium sulfate (20%) were added slowly until precipitation could be seen.

After some minutes of resting the suspension was centrifuged at 20000 rpm for 15 min. The pellet was then resuspended in 2 mL of standard saline citrate buffer. After taking a sample of 80 µL the 4 samples ware mixed with 20 µL SDS-loading buffer each and analysed on an SDS-PAGE.

The purified filaments were frozen in liquid nitrogen and then at -80°C.

The flagella purification was successful. Besides the flagellin the samples also contain the hook proteins, the smaller proteins were removed from the purified pellet.

14.69 tRNAPhe assay

Aim: Measure aminoacylation levels

To measure aminoacylation of tRNAPhe the components were mixed.

Content Final concentration
Dialysis-buffer ad 250 µL
DTT 1 mM
MgCl2 20 mM
KCl 10 mM
tRNAPhe 10 µM
L- or D-Phenylalanine 1 mM
catalytic fusion Protein 10 µM

After mixing, reaction was initiated by the addition of 2 mM ATP and then incubated for 1 to 60 min by 37 °C and 350 rpm. Assays were stopped by the addition of 25 µL 3 M sodium acetate solution. Afterwards tRNAs were precipitated with ethanol. Dissolved pellets were further purified using size-exclusion columns. The elution fraction was split into two aliquots. One samples was treated with 0.2 M NaOH (final concentration: 10 mM) and incubated at 50 °C for 10 min, while the other aliquot was kept on ice. The base treated sample was again acidified by the addition of 3 M sodium acetate (pH 5.2, final concentration: 300 mM). The volume of the untreated sample was adjusted using 2 mM sodium acetate (pH 5.2). Subsequently, both samples were subjected to LCMS analysis using 100 µL for injection.

11.08.2014

25. Cell culture assays
25.1 Splitting of Caco-2-cells

Aim: Splitting cell culture for growing

The Caco-2-cells were kindly provided by Dr. Hartmann Reifert (BMFZ). In order to let them grow, the cells had to be splitted according to a specific protocol. Caco-2-cells require DMEM (Dulbecco’s Modified Eagle Medium) with 20% FCS and L-Glutamine.

A centrifuge was precooled to 4°C.

The medium of the culture flask was discarded from the side opposite to the adherent cells and washed with 1X PBS from the opposite site as well.

The PBS was aspirated with a glass pipette until the supernatant was clear. After adding 1 - 2 mL 1x trypsin from the opposite of the adherent cells the flask was incubated for 5 - 10 min at 37°C depending on how fast the cells unfastened of from the ground. Under the microscope the free floating cells were checked.

The cells were transferred into a 15 mL falcon and rinsed with 10 mL DMEM to collect all remaining cells in the flask. The 15 mL falcon was filled with DMEM and the culture centrifuged at 1500 rpm  for 5 min at 4°C.

The supernatant was discarded and resuspended in 2 mL DMEM. The cells were splitted 1:3 which means that 666 µL were taken from the suspension and transferred into a new culture flask. After adding 20-25 mL DMEM the flask was incubated at 37°C and checked on the 3rd / 4th day under the microscope.

On the medium flask were the following notes:
Date, Name, Cell Line, Medium, Ratio of splitting, passage number

15.63 PCR Amplification of Hag-D2-Cup

Aim: Amplification of insert for Gibson assembly into piGEM-005

The synthesized fragments by Integrated DNA technologies arrived as a dried 200 ng DNA pellet. In order to use it as a PCR template the pellet was centrifuged and resuspended in 20 µL Millipore water to get an end concentration of 10 ng/µL as a Stock solution.

0,5 µL were used as a PCR template.

Mix (µl) Mastermix D2-Cup D2-Strep
Template - 2 2
Primer Flo96 D23-fw 3 - -
Primer Flo97 D23-rv 3 - -
Q5-Master mix 75 - -
Water 63 - -
Mastermix - 48 48
Total Volume 144 50 20

Step Temperature °C Time
1 98 3 min
2 98 30 sec
3 55 30 sec
4 72 30 sec
5 Go To 2 31x
6 72 4min
7 8 infinite


The expected bands could be seen.

15.64 Gibson assembly with linearized piGEM-005 & PCR products from 15.63

Aim: Insertion of Hag-D2-Cup and Hag-D2-Strep into piGEM-005

2
Component Hag-D2-Cup(µL) Hag-D2-Strep (µL)
Gibson-mix 15 15
Insert I:
 (Hag-D2-Cup 431 ng/µL)
2,5 -
Insert II:
(Hag-D2-Strep 423 ng/µL)
- 2,5
piGEM-005 SpeI
 (50 ng/ µL)
2,5 2,5
Total 20 20

Incubation was done  at 50°C for 1 hour with following transformation into E. coli XL1-Blue plated out on LB-Amp plates and incubated overnight at 37°C.

12.08.2014

15.64 Screening clones from 15.63

Aim: Checking insertion of Hag-D2-Cup and Hag-D2-Strep into piGEM-005

In order to check the successful integration of D2-Cup and D2-Strep, clones were picked for cPCR and transferred onto a LB-Amp plate. The primers Flo96 (D23-fw) and Flo97 (D23-v) were used.

Mix (µl) Mastermix D2-Cup D2-Strep
Template - Colony Colony
Primer Flo96 D23-fw 4,5 - -
Primer Flo97 D23-rv 4,5 - -
dNTP 4,5 - -
5x Phusion Buffer 36 - -
Phusion DNA-polymerase 4,5 - -
Water 126 - -
Mastermix - 20 20
Total Volume 180 20 20

Step Temperature °C Time
1 98 10 min
2 98 30 sec
3 55 30 sec
4 72 40 sec
5 Go To 2 33x
6 72 4min
7 8 infinite

The gel shows that clone 2 from the Gibson Hag-D2-Cup clones and clones 3 & 4 from the Gibson Hag-D2-Strep clones contain the integrated plasmid constructs. All 3 clones were picked for a miniprep and used for the inoculation of 10 mL LB-Amp. Incubation was performed over night at 37°C.

The new constructs were labelled with piGEM-026 (pMAD-Hag-D2-Cup) and piGEM-027 (pMAD-Hag-D2-Strep).

15.65 Overnight culture for WT3610

Aim:Preparation for mainculture the next day

5 mL of LB were inoculated with Bacillus subtilis WT3610 from an LB plate and incubated at 37°C over night.

13.08.2014

15.66 Transformation of competent Bacillus subtilis WT3610

Aim: transformation of plasmid into Bacillus subtilis WT3610

100 µL preculture were added to 10 mL of MNGE-Medium and incubated to an OD600 of 1,1-1,3 at 37°C which could take 4-5h.

After reaching an OD600 of 1,1-1,3 2 x 400 µL of the culture were transformed with 1,5 µg plasmid (piGEM-026 & -027). After 1 hour of incubation at 37°C 100 µL expression mix were added and incubated for 1 hour as well.

In the end the 500 µL attempt was plated out on MLS-X-Gal plates and incubated at 30 °C overnight until colonies could be seen.

Sequencing
Premix Label-Nr. Construct Primer
1 AGB0023458 piGEM-023 TW260
2 AGB0023459 piGEM-024 TW260
3 AGB0023460 piGEM-025 TW260
4 AGB0023461 piGEM-026 D2-Cup cl.2 Flo96-D2 3 for
5 AGB0023462 piGEM-026 D2-Cup cl.2 Flo56-Hag rv
6 AGB0023463 piGEM-027 D2-Strep cl.3 Flo96-D2 3 for
7 AGB0023464 piGEM-027 D2-Strep cl.3 Flo56-Hag rv
8 AGB0023465 piGEM-027 D2-Strep cl.4 Flo96-D2 3 for
9 AGB0023466 piGEM-027 D2-Strep cl.4 Flo56-Hag rv
13.72 Restriction of Nose plasmids with EcoRV

Aim: check if amyE and cat are still in plasmid, since B. subtilis could not successfully be transformed with the plasmids yet

All Nose-plasmids (iGEM002-004 and 007-015) were digested with EcoRV to check if amyE and cat are still present in these plasmids, since B. subtilis could not be transformed successfully.

Per sample 1 µl 2x CutSmart Buffer, 7.9 µl H2O, 0.1 µl EcoRV and 2 µl of plasmids were added. The reaction was incubated over night at RT.

14.08.2014

13.72 Restriction of Nose plasmids with EcoRV - continuation

Aim: Analysis of the restriction on an agarose gel

The samples were mixed with 2 µl of 5x Loading dye and 8 µl of it was loaded on a 1% agarose gel.

All plasmids but piGEM004 and piGEM011 show the expected bands of ca 2000 and 4000 bp.

The plasmids were used to transform Bacillus subtilis, which was incubated at 30°C.

18.08.2014

15.67 Transformation of competent WT3610

Aim: Bacillus subtilis pMad Trafo mit D2-Strep and D2-Cup

Next step: inoculate overnight culture from the X-gal plate with 3 ml LB-MLS.

As the cultures were recognizable blue (thin or old plate) wildtype cultures were inoculated for a new transformation. Therefore the plasmids piGEM-026 and -027 were isolated from frozen pellets.

19.08.2014

15.68 Transformation of competent WT3610

Bacillus subtilis pMad trafo mit D2-Strep and D2-Cup

For the experimental procedure see the Materials section. Anyway, two transformations were running currently that will be referred to as 'old' and 'new' transformation from now on.

For the old transformation the first temperature shift was performed in LB-MLS medium. The culture was plated on LB-MLS-X-Gal plates and incubated at 42°C over night.

The new transformation was started with a culture grown in MNGE medium, inoculated with a wildtype overnight culture. The culture was grown until an optical density of 1,2 that is normally reached after 4-5 h. Strangely the culture reached this OD after almost 7 h today. Plasmids (1.5µg) piGEM026 and -027 were transformed and the protocol was followed as described below.

20.08.2014

15.69 Transformation of competent WT3610

Bacillus subtilis pMad Trafo mit D2-Strep and D2-Cup

New trafo showed no colonies so far. It was further incubated.

For the old trafo 2nd temperature shift of positive colonies (only light blue).

21.08.2014

15.70 Transformation of competent WT3610

Bacillus subtilis pMad Trafo mit D2-Strep and D2-Cup

Old trafo: white colonies were re-streaked on x-Gal plates and on MLS plates to check for MLS sensitivity. Incubation at 42°C.

22.08.2014

15.70 Transformation of competent WT3610

Aim: integration of pMAD-Insert into Bacillus chromosome via flanks

The blue/ white screening showed positive transformed blue clones from the new transformation.  Three piGEM026 and -027 clones of different morphology per plate were picked and used for inoculation of LB-MLS (4 mL LB, 4 µL lincomycin, 4 µL erythromycin). Incubation was carried out over night at 30°C with the cultures.

22. The Killswitch
22.1 Amplification of flanks

Aim: The flanks have to be amplified for a fusion PCR with the Killswitch modules in order to integrate them into the amyE and the lacA/ganA locus of Bacillus subtilis after cloning into pMAD

Primers arrived and have been dissolved in millipore water, dilution 1:10. Q5 Master Mix was used for the following PCRs to create the flanks of the killswitch parts I-III:

Fragment Fw primer (iGEM-0xx) Rv primer (iGEM-0xx)
AmyE flankI iGEM-034 iGEM-035
AmyE flankII iGEM-036 iGEM-037
lacA flankI iGEM-040 iGEM-041
lacA flankII iGEM-042 iGEM-043
KSIII AmyE-yvyd FlankII iGEM-048 -

Mix Master Mix amyE FlankI amyE FlankII lacA FlankI lacA FlankII amyE-yvyD FlankII
Template (B. subtilis gDNA) 11 - - - - -
Primer iGEM-34 - 1 - - - -
Primer iGEM-35 - 1 - - - -
Primer iGEM-36 - - 1 - - -
Primer iGEM-37 - - 1 - - -
Primer iGEM-40 - - - 1 - -
Primer iGEM-41 - - - 1 - -
Primer iGEM-42 - - - - 1 -
Primer iGEM-43 - - - - 1 -
Primer iGEM-48 - - - - - 1
Primer iGEM-37 - - - - - 1
Q5-Master mix 165 - - - - -
Water 132 - - - - -
Mastermix - 28 28 28 28 28
Total Volume [µl] 308 30 30 30 30 30

Step Temperature °C Time
1 98 3 min
2 98 30 sec
3 55 30 sec
4 72 60 sec
5 Go To 2 30x
6 72 4min
7 8 infinite

All bands had the expected size (500 /1000 bp)

23.08.2014

15.71 Transformation of competent WT3610

Aim: First heat shock - integration of pMAD-Insert into Bacillus chromosome via flanks

The  overnight cultures were used to inoculate 10 mL LB MLS until  the culture obtained an OD of 0,1. The cultures were incubated at 30°C for 2h.

Then the temperature was shifted to 42°C for 6h.

After the heat shock dilutions from 10-5 to 10-6 of each culture were plated out on MLS-X-Gal so that plates could be incubated overnight at 42°C.

24.08.2014

22.2 Amplification of Killswitch modules I-III

Aim: The modules had to be amplified via PCR in order to proceed with a fusion PCR linking the modules with the flanks for integration into the Bacillus Loci.

The synthesized fragments by Integrated DNA technologies arrived as a dried 200 ng DNA pellet. In order to use it as a PCR template the pellet was centrifuged down and resuspended in 20 µL millipore water to get an end concentration of 10 ng/µL as a stock solution.

0,5  µL were used as a PCR template. The PCR fragments should be ca. 350, 650 & 2000 bp long.

Mix KS module I KS module II KS module III
Template (gBlock DNA) 0,5 0,5 0,5
Primer iGEM-38 1 - -
Primer iGEM-39 1 - -
Primer iGEM-44 - 1 -
Primer iGEM-45 - 1 -
Primer iGEM-49 - - 1
Primer iGEM-50 - - 1
Q5-Mastermix 25 25 25
Water 22,5 22,5 22,5
Total Volume (µl) 50 50 50

q
Step Temperature °C Time
KSI & III
Time
KSII
1 98 3 min 3 min
2 98 20 sec 30 sec
3 55 20 sec 20 sec
4 72 60 sec 120 sec
5 Go To 2 31x 31x
672 4min 4min
7 8 infinite infinite


The modules I and III could be amplified without problems, but KSII showed no distinct band and many unspecific fragments.

15.71 Transformation of competent WT3610

Aim: Second heat shock -flip out of the pMAD backbone

One blue colony per diluted piGEM026 and -027 clone was used to inoculate 4 mL LB. The cultures were incubated at 30°C for 6 hours and afterwards for 3 hours at 42°C.

Dilutions from 10-5 to 10-6 were plated out on  X-Gal plates without MLS selection. The positive clones should not contain the resistance inside the backbone as well as the galactosidase. The plates were incubated at 42°C overnight. Some clones (piGEM027.1 and one of 27.2) did not grow and will be inoculated again. They will be inoculated in LB-MLS for the first temperature shift tomorrow.

22.3 Transformation of competent DH5α with pMAD

Aim: Gain E.coli on plate for mini preps of pMAD

Standard heat shock transformation protocol was followed, incubation over night at 37°C. pMAD from the plasmid box was used for transformation.

22.4 Restriction of pMAD with NcoI and BamHI over night

Aim: Linearizing Vector for Gibson Assembly with KS modules

Component Volume (µl)
pMAD (108 ng/µL) 9
CutSmart 10x 2
NcoI 0,2
BamHI 0,2
Water 8,6
Total Volume 20

Incubation over night at room temperature.

25.08.2014

15.72 Transformation of competent WT3610

Aim: checking the correct flip out of the pMAD backbone and MLS sensitivity

From the dilution plates was one white clone picked and transferred on a X-Gal plate as well as on a MLS plate so that clones were proven for the right integration of the insert although flipping out the pMAD backbone.

The plates were incubated at 42°C overnight.

22.5a Fusion PCR

Aim: Assembly of Killswitch modules I & II with amyE and lacA flanks

Segment Concentraction (ng/µL)
KS module I 401
KS module II 414
KS module III 369
amyE flank I 446
amyE flank II 403
lacA flank I 368
lacA flank II 369
amyE yvyD flank II 372

Mix amyE KS I lacA KS II
KS module I (40,1 ng/µL) 1 -
KS module II (41,4 ng/µL) - 1
amyE flank I ( 44,6 ng/µL) 1 -
amyE flank II (40,3 ng/µL) 1 -
lacA flank I (36,8 ng/µL) - 1
lacA flank II (39,6 ng/µL) - 1
Primer iGEM-34 1 -
Primer iGEM-37 1 -
Primer iGEM-40 - 1
Primer iGEM-43 - 1
Phusion Buffer 5x 10 10
Phusion DNA-polymerase 1 1
dNTPs 1 1
Water 33 33
Total Volume [µl] 50 50

q
Step Temperature °C Time
amyE KSI
Time
amyE KSII
1 98 3 min 3 min
2 98 20 sec 30 sec
3 55 20 sec 20 sec
4 72 120 sec 190 sec
5 Go To 2 31x 31x
672 4min 4min
7 8 infinite infinite

No PCR amplificates could be seen on the gel.

18.53 PCR amplification of StrepDARPidin

Aim: amplification of the StrepDARPidin for cloning into the expression vector pET24d for protein purification and overproduction in E. coli BL21(DE3)

The synthesized fragments by Integrated DNA technologies arrived as a dried 200n g DNA pellet. In order to use it as a PCR template the pellet was centrifuged and resuspended in 20 µL millipore water to get an end concentration of 10 ng/µL as a stock solution.

0,5  µL were used as a PCR template. The PCR fragment should be ca. 900 bp long.

Mix PCR attempt
Template 0,5
Primer iGEM-33 1
Primer iGEM-34 1
Q5-Mastermix 25
Water 22,5
Mastermix -
Total Volume 50

Step Temperature °C Time
1 98 3 min
2 98 30 sec
3 55 30 sec
4 72 60 sec
5 Go To 2 31x
6 72 4min
7 8 infinite

Both samples had a concentration of 380 ng/µL.

18.54 Digestion of amplified of StrepDARPidin for cloning into pET24d

Aim: digest with NcoI and XhoI of the StrepDARPidin PCR product for cloning into the expression vector pet24d

The PCR product was cut with NcoI and XhoI to get the restriction sites for cloning into pET24d NcoI/XhoI cut.

Component StrepDARPidin (µl)
PCR product (380 ng/µL) 6
CutSmart 10x 2
NcoI 0,25
XhoI 0,25
Water 11,5
Total Volume 20

Incubation for 1h at 37°C with heat shock at 85°C for 10 min.

18.55 Ligation of digested StrepDARPidin for cloning into pET24d

Aim: ligation of StrepDARPidin PCR product with the expression vector pET24d

The PCR product was ligated into digested pET24d NcoI/XhoI. Two samples were made for a transformation of E. coli XL1-Blue and BL21.

Component Ligation I (µl) Ligation II (µl)
NcoI and XhoI StrepDARPidin (64 ng/µL) 2 2
pET24d NcoI and XhoI (15 ng/µL) 5 5
Ligation Buffer 10x 2 2
Ligase 1 1
Water 10 10
Total Volume 20 20

Incubation for 1,5h at room temperature with heat shock at 85°C for 10 min. The 20 µL from ligation I were used to transform E. coli XLI-Blue, 10 µL from Ligation II were used to transform E. coli BL21(DE3) and the rest of ligation II into Xl1-Blue as well and selected on LB-Canamycin plates.

26.08.2014

15.73 Transformation of competent WT3610

Aim: checking the correct flip out of the pMAD backbone and MLS sensitivity

All clones on the master plate grew on x-gal plates but were MLS sensitive and could be seen as positive. In order to check the correct insertion of D2-Cup an D2-Strep into the Bacillus subtilis genome the clones were cooked in 100 µL 1x PBS at 95°C for 10 min and used for colony PCR. Because of the repetition of the temperature shifts with clones 27.1.1, 27.1.2, 27.1.3 yesterday and today and 27.2.3, 6 clones from 26.1 -26.3, 27.2.1 & 27.2.2 were picked for the cPCR (30 reactions).

Mix (µl) Master-Mix for 30 attempts (µL) Mix for PCR attempt (µL)
Colony PBS suspension - 2
Primer Flo54 Hag-fw 15 -
Primer Flo56 Hag-fw 15 -
Phusion DNA-polymerase 15 -
Phusion Buffer 5x 120 -
dNTP-mix 15 -
Water 410 -
DMSO 10 -
Mastermix - 18
Total Volume 600 20

Step Temperature °C Time
1 98 3 min
2 98 30 sec
3 55 30 sec
4 72 1 min 30 sec
5 Go To 2 30x
6 72 4min
7 8 infinite

There were no bands visible on the gel.

18.56 cPCR with plates from ligation

Aim: checking the success of the ligation

The grown clones from the ligation the day before were inoculated for plasmid isolation and then checked via cPCR with primers iGEM-032 and -033.

Content (µl) Master mix  for 20 clones (µL) Clones from Ligation I (µL) Clones from Ligation II (µL) Clones from Ligation III (µL)
Template (colony) - 1 1 1
Primer iGEM-32 10 - - -
Primer iGEM-32 10 - - -
Phusion Buffer 5x 80 - - -
Phusion DNA-polymerase 10 - - -
dNTP-mix 10 - - -
Water 280 - - -
Mastermix - 19 19 19
Total Volume 400 20 20 20

Step Temperature °C Time
1 98 10 min
2 98 20 sec
3 55 20 sec
4 72 60 sec
5 Go To 2 33x
6 72 4min
7 8 infinite

The gel shows that the clones I 2-6, II 1-6 and III 2, 3, 4 and 6 are positive due to the band at approx. 900 bp. The other bands might be plasmid which was used as template.

The clones I6, II3, III3 were used to transform E. coli BL21(DE3) and selected on LB-Can plates. III4 and III6 were already on a plate. The clones were inoculated for plasmid isolation and sent for sequencing.

22.5b Fusion PCR Attempt 3

Aim: Fusion of amyE and lacA flanks to the killswitch fragments

The Fusion PCR was repeated since it did not work the first two times. The fragments of the killswitch I and II were diluted 10fold. A gradient was carried out from 52°C to 62°C.

Mix (µl) amyE KSI lacA KS II
KS module I (40,1 ng/µL) 1 -
KS module II (41,4 ng/µL) - 1
amyE flank I ( 44,6 ng/µL) 1 -
amyE flank II (40,3 ng/µL) 1 -
lacA flank I (36,8 ng/µL) - 1
lacA flank II (39,6 ng/µL) - 1
Primer iGEM-34 1 -
Primer iGEM-37 1 -
Primer iGEM-40 - 1
Primer iGEM-43 - 1
Phusion Buffer 5x 4 4
Phusion DNA-polymerase 0,5 0,5
dNTPs 1 1
Water 9,5 9,5
Total Volume 20 20

Step Temperature °C Time
1 95 5 min
2 95 30 sec
3 52-62 30 sec
4 72 200 sec
5 Go To 2 32x
6 72 5 min
7 4 infinite

Again, no fragments could be noticed on the gel.

13.73 Amplification of new Cu- and Ag-Promotor sequences

Aim: Amplification of Cu- and Ag promoter sequences from the chromosomal DNA of Bacillus subtilis.

Content Master mix  (µL) 2x Attempt Cu (µL) 2x Attempt Ag (µL)
Template (gDNA B. subtilis) 4 - -
Primer iGEM-55 - - 1
Primer iGEM-56 - - 1
Primer iGEM-57 - 1 -
Primer iGEM-58 - 1 -
Phusion Buffer 5x 40 - -
Phusion DNA-polymerase 4 - -
dNTPs 4 - -
Water 144 - -
Mastermix - 48 48
Total Volume (µl) 196 50 50

Step Temperature °C Time
1 98 10 min
2 98 20 sec
3 55 20 sec
4 72 20 sec
5 Go To 2 30x
6 72 4 min
7 8 infinite

The gel shows positive bands at approx. 100 bp and 250 bp, which was like expected.

13.74 NcoI/SacI digest of piGEM-002

Aim: linearizing the plasmid backbone for Gibson Assembly and Ligation with Cu/Ag promotor sequences and IPTG/ Constitutive (/tetO) promotor sequences.

Component Attempt I (µl)
piGEM-002 (476 ng/µL) 6,5
NcoI 0,25
SacI 0,25
CutSmart Buffer 10x 2
Water 11
Total Volume 20

Incubation at 37°C for 60 min and heat shocked at 85°C for 10 min.

13.75 Ligation of NcoI/Sac cut piGEM-002 with annealed oligos containing promoter sequences

Aim: insertion of a IPTG inducible/ constitutive and constitutive+tetO into piGEM-002

In order to test our nose system with new promoter sequences oligos were designed and annealed by heating up water and letting it cool down with the oligo pairs iGEM-59/60, -61/62, - 63/64 so that the individual fragments were able to anneal with overlaps to the NcoI/SacI sites.

Component Ligation I (µl) Ligation II (µl) Ligation III (µl)
piGEM-002 (209 ng/µL) 5 5 5
iGEM-59/60 1 - -
iGEM-61/62 - 1 -
iGEM-63/64 - - 1
T4 Ligase 1 1 1
Ligation Buffer 10x 2 2 2
Water 11 11 11
Total Volume (µl] 20 20 20

The reactions were incubated at room temperature for an hour and transformed into E. coli XL1-Blue, plated out on LB-Amp.

13.76 Gibson assembly of NcoI/SacI piGEM-002 & Cu/ Ag promotor

Aim: assembling of Nco/Sac cut piGEM-002 with Cu/ ag promotor sequences

In order to test new Cu/ Ag promotor sequences the PCR fragments from 13.73 were used for a Gibson assembly.

Component Gibson Cu I (µL) Gibson Ag II (µL) Control  (µL)
piGEM-002 (209 ng/µL) 2,5 2,5 5
Cu promotor 2,5 - -
Ag promotor - 2,5 -
Gibson mix 15 15 15
Total Volume 20 20 20

The attempts were incubated at 50°C for an hour and transformed into E. coli XL1-Blue, plated out on LB-Amp.

27.08.2014

22.3b Repeated Amplification of Killswitch modules I and II

Aim: Increase the amount of modules for further purification, since Fusion PCR did not work 'quick and dirty'

This time the Phusion polymerase was used. The template was the already amplified module.

Mix KS module I KS module II
Template (already amplified modules) 0,5 0,5
Primer iGEM-38 1 -
Primer iGEM-39 1 -
Primer iGEM-44 - 1
Primer iGEM-45 - 1
5x HF buffer 10 10
dNTPs 1 1
Water 36 36
Phusion DNA-polymerase 0,5 0,5
Total Volume (µl) 50 50

Step Temperature °C  KS I KS II
1 98 5 min 5 min
2 98 30 sec 30 sec
3 55 30 sec 30 sec
4 72 120 sec 200 sec
5 Go To 2 34x 34x
6 72 5 min 5 min
7 8 infinite infinite

The gel can be seen under the next topic

22.3c Repeated amplification of killswitch flanks

Aim: The flanks have to be amplified for a fusion PCR with the Killswitch modules in order to integrate them into the amyE and the lacA locus of Bacillus subtilis after cloning into pMAD, yield higher amount of fragments for purification and FusionPCR.

The Phusion polymerase was used.

Mix Master Mix amyE FlankI amyE FlankII lacA FlankI lacA FlankII amyE-yvyD FlankII
Template   0,5 - - - -
Primer iGEM-34 - 1 - - - -
Primer iGEM-35 - 1 - - - -
Primer iGEM-36 - - 1 - - -
Primer iGEM-37 - - 1 - - -
Primer iGEM-40 - - - 1 - -
Primer iGEM-41 - - - 1 - -
Primer iGEM-42 - - - - 1 -
Primer iGEM-43 - - - - 1 -
Primer iGEM-48 - - - - - 1
Primer iGEM-37 - - - - - 1
dNTPs 11 1 - - - -
DMSO 3,3 0,3        
MgCl2 2,2 0,2        
5x HF-buffer 110 10        
Phusion DNA-polymerase 5,5 0,5        
Water 390,5 35,5        
Mastermix - 47,5 47,5 47,5 47,5 47,5
Total Volume (µl) 522,5 50 50 50 50 50


With exception of KSI and StrepDARP the PCR was negative for all other samples. Even for the positive reactions the bands were very thin.

18.57 repeated PCR amplification of Hag-KpnI StrepDARPidin

Aim: amplification of the StrepDARPidin for cloning into the expression vector pET24d for protein purification and overproduction in E. coli BL21(DE3)

The synthesized fragments by Integrated DNA technologies arrived as a dried 200 ng DNA pellet. In order to use it as a PCR template the pellet was centrifuged and resuspended in 20 µL millipore water to get an end concentration of 10 ng/µL as a stock solution.

0,5  µL were used as a PCR template. The PCR fragment should be approx. 900 bp long.

Content (µl) 2x Attempt I (µL) 2x Attempt II(µL)
Template 1: StrepDARPidin (38 ng/ µL) - 1
Template 2: Hag-KpnI (10 ng/ µL) 1 -
Primer iGEM-51 - 1
Primer iGEM-52 - 1
Primer iGEM-53 1 -
Primer iGEM-54 1 -
Phusion Buffer 5x 10 10
Phusion DNA-polymerase 1 1
dNTPs 1 1
Water 35 35
Total Volume 50 50

Step Temperature °C Time
1 98 3 min
2 98 30 sec
3 55 30 sec
4 72 60 sec
5 Go To 2 31x
6 72 4min
7 8 infinite


The PCR was clearly positive for StrepDARP, Hag-KpnI could be seen as a very slight band at 900 bp, but more unspecific bands could be noticed.

18.56 Expression-Test with transformed E. coli BL21(DE3)

Aim: Checking the overexpression of pET24d-StrepDARPidin

In order to check the expression of the proteins 5 clones (I6, II3, III3, III4, III6) containing piGEM-028 (pet24d-StrepDARPidin) were used to inoculate 20 mL of LB-Can. 5 cultures were incubated at 37°C until an OD of 0,7.

A ca. 1 mL preinduction sample (PI) was taken (0,7 (favoured OD) : 0,7 (actual OD) = 1 mL). After that the cultures were induced with 20 µL IPTG for 2h.

An induction sample (I) was taken (320 µL with an OD of 2,2; 350 µL with an OD of 2).

PI and I were centrifuged at 14000 rpm for 1 min, the supernatant was discarded and the pellet resuspended in 60 µL water and 40 µL SDS-Buffer.

The four samples were analysed on an SDS-PAGE gel with 10 µL volume per sample.

The gel did not show any signs of over expression so that lactose was chosen as an alternative inducer to test the expression. New test expression cultures (I6, II3, III3, III4, III6)  were made with 100 mL LB-Can and 5 mL lactose (6,25 g/ 25 mL) per culture. Incubation was performed over night at 30°C.

28.08.2014

Sequencing of constructs from 28.08.2014
Premix Label-Nr. Construct Primer
1 AGB0023467 piGEM-028 I6(pET24d-StrepDARPidin) T7 turn
2 AGB0023468 piGEM-028 III3 (pET24d-StrepDARPidin) T7 turn
3 AGB0023469 piGEM-026 (pMAD-D2-Cup) Flo D2_3 fw
4 AGB0023470 piGEM-027.1 (pMAD-D2-Strep) Flo D2_3 fw
5 AGB0023471 piGEM-027.2 (pMAD-D2-Strep) Flo D2_3 fw
18.56 Expression-Test with transformed E. coli BL21(DE3)

Aim: Checking the overexpression of pET24d-StrepDARPidin


No sign of an overexpression could be noticed.

18.57 His-bead purification of pellet from E. coli BL21(DE3)

Aim: Checking the overexpression of pET24d-StrepDARPidin

The 100 mL culture from I6 & III3 were centrifuged at 4000 rpm for 10 min in 2x50 mL falcons. The pellets were washed in 10 mL buffer A and cracked with the microfluidizer. After that the suspension was centrifuged at 20000 rpm for 20 min/ 4°C. The supernatant was transferred into a 50 mL falcon.

200 µL Ni-NTA beads were added to the suspension, inverted and incubated on ice for half an hour.

After that the suspension was centrifuged or 5 min at 4000 rpm so that the pellet could be resuspended in 500 µL Buffer A after discarding the supernatant. The 500 µL were transferred into a 1,5 mL tube and centrifuged at 4000 rpm for washing. After discarding the supernatant the pellet was washed again with 500 µL Buffer A. After a second centrifugation at 4000 rpm the supernatant was discarded, the pellet resuspended in 200 µL Buffer A and centrifuged at 13000 rpm to destroy the Ni-bead – protein interaction.

In the end the supernatant was transferred into a new 1,5 mL tube. The Ni-beads were mixed with 100 µL 20% ethanol.

40 µL of the supernatant from clone I6 and III3 were added with 10 µL SDS-PAGE Buffer and analysed on the SDS-Gel (visible at 18.56).

22.3d Repeated amplification of killswitch flanks

Aim: The flanks have to be amplified for a fusion PCR with the Killswitch modules in order to integrate them into the amyE and the lacA locus of Bacillus subtilis after cloning into pMAD, yield higher amount of fragments for purification and FusionPCR.

Mix [µl] Master Mix 6x amyE FlankI amyE FlankII lacA FlankI lacA FlankII amyE-yvyD FlankII
Template (B. subtilis gDNA) 6 - - - - -
Primer iGEM-34 - 1 - - - -
Primer iGEM-35 - 1 - - - -
Primer iGEM-36 - - 1 - - -
Primer iGEM-37 - - 1 - - -
Primer iGEM-40 - - - 1 - -
Primer iGEM-41 - - - 1 - -
Primer iGEM-42 - - - - 1 -
Primer iGEM-43 - - - - 1 -
Primer iGEM-48 - - - - - 1
Primer iGEM-37 - - - - - 1
Q5-Master mix 150 - - - - -
Water 132 - - - - -
Mastermix - 48 48 48 48 48
Total Volume 288 50 50 50 50 50

Step Temperature °C Time
1 98 5 min
2 98 25 sec
3 55 25 sec
4 72 60 sec
5 Go To 2 31x
6 72 4 min
7 8 infinite

The gel is visible under 22.3e

22.3e Repeated Amplification of Killswitch modules I and II

Aim: Increase the amount of modules for further purification, since Fusion PCR did not work 'quick and dirty'. Q5 was used and gDNA since the previously amplified modules did not work as template yesterday.

Mix [µl] KS module I KS module II KS module III
Template (gBlock DNA) 0,5 0,5 0,5
Primer iGEM-38 1 - -
Primer iGEM-39 1 - -
Primer iGEM-44 - 1 -
Primer iGEM-45 - 1 -
Primer iGEM-49 - - 1
Primer iGEM-50 - - 1
Q5-Master mix 25 25 25
Water 2,5 2,5 2,5
Total Volume 50 50 50

Step Temperature °C  KS I & III KS II
1 98 5 min 5 min
2 98 25 sec 25 sec
3 55 25 sec 25 sec
4 72 60 sec 120 sec
5 Go To 2 31x 31x
6 72 4 min 4 min
7 8 infinite infinite

The expected bands could be seen for the flank I of amyE and flank I and II of lacA. These bands were cut out and purified via a Gel Extraction kit, but the concentration was too low to work further.

13.75 NcoI/SacI digest of piGEM-002

Aim: linearizing the plasmid backbone for Gibson Assembly and Ligation with Cu/Ag promotor sequences and IPTG/ Constitutive (/tetO) promotor sequences.

The already digested piGEM002 of 26.08. was nearly empty. To have some digested plasmid as backup a new digestion was carried out. piGEM002 was isolated and used for the restriction.

Component Attempt (µl)
piGEM-002 (476 ng/µL) 10
NcoI 0,25
SacI 0,25
CutSmart Buffer 10x 2
Water 7,5
Total Volume 20


An agarose gel of the previous restriction was done with unrestricted piGEM002 as reference. A band at ca between 6000 and 8000 bp could be seen, which indicates a successful restriction (expected size: ca 6600 bp).

13.76 Repeated Gibson assembly of NcoI/SacI piGEM-002 & Cu/ Ag promotor

Aim: assembling of NcoI/SacI cut piGEM-002 with Cu/Ag promoter sequences

In order to test new Cu/ Ag promoter sequences the PCR fragments from 13.73 were used for a Gibson assembly.

Component Gibson Cu I (µL) Gibson Ag II (µL) Control  (µL)
piGEM-002 (ca. 40ng/µL) 2,5 2,5 2,5
Cu promotor (ca 4 ng/µl) 2,5 - -
Ag promotor (ca 4 ng/µl) - 2,5 -
H2O - - -
Gibson Mix 15 15 15
Total Volume 20 20 20

The attempts were incubated at 50°C for an hour and used to transform E. coli XL1-Blue, plated out on LB-Amp.

13.77 Repeated ligation of NcoI/SacI cut piGEM-002 with annealed oligos containing promoter sequences

Aim: insertion of an IPTG inducible/ constitutive and constitutive+tetO into piGEM-002

The ligation was prepared to contain ca 100 ng of the restricted piGEM002 with an appropriate amount of insert (much fewer).

Component Ligation I (µL) Ligation II (µL) Ligation III (µL)
piGEM-002 (ca 40 ng/µL) 2,5 2,5 2,5
iGEM-59/60 (ca 4 ng/µl) 0,8 - -
iGEM-61/62 (ca 4 ng/µl) - 0,8 -
iGEM-63/64 (ca 4 ng/µl) - - 0,8
Polynucleotide kinase 1 1 1
T4 Ligase 1 1 1
Ligation Buffer 10x 2 2 2
Water 12,7 12,7 12,7
Total Volume 20 20 20

The reactions were incubated at room temperature for an hour and transformed into E. coli XL1-Blue, plated out on LB-Amp.

29.08.2014

Sequencing results of constructs from 28.08.2014
Premix Label-Nr. Construct Results
1 AGB0023467 piGEM-028 I6(pET24d-StrepDARPidin) No results
2 AGB0023468 piGEM-028 III3 (pET24d-StrepDARPidin) No alignment
3 AGB0023469 piGEM-026 (pMAD-D2-Cup) Good
4 AGB0023470 piGEM-027.1 (pMAD-D2-Strep) Point mutation
5 AGB0023471 piGEM-027.2 (pMAD-D2-Strep) Point mutation

The sequencing showed that pET24d did not contain any known insert.

In order to check the correct synthesis of the templates purified PCR products were sent for sequencing again. In the meanwhile the constructs were cloned again.

Sequencing of constructs from 29.08.2014

Premix Label-Nr. Construct Primer
1 AGB0023512 PCR Fragment StrepDARPidin I piGEM-032 fw
2 AGB0023513 PCR Fragment StrepDARPidin II piGEM-032 fw
3 AGB0023514 PCR Fragment D2-StrepII Flo96 D2_3 fw
4 AGB0023515 PCR Fragment StrepDARPidin I piGEM-033 rv
5 AGB0023516 PCR Fragment StrepDARPidin II piGEM-033 rv
6 AGB0023517 PCR Fragment D2-StrepII Flo97 D2_3 rv
18.58 New PCR amplification of StrepDARPidin and D2-Cup

Aim: amplification of the StrepDARPidin for cloning into the expression vector pET24d for protein purification and overproduction in E. coli BL21(DE3) & amplification of D2-StrepII for Gibson Assembly into piGEM-005

The synthesized fragments were supposed to be sequenced in order to check the right synthesis.  10 ng/µL as a Stock solution and a purified PCR Product were used as template for each reaction.

0,5  µL were used as a PCR template. The PCR fragment should be ca. 900 bp long for StrepDARPidin and ca. 450 bp for D2-StrepII.

Mix (µl) PCR StrepDARPidin I PCR StrepDARPidin II PCR StrepDARPidin III PCR D2-Strep I PCR D2-Strep II
Pur. StrepDARP. (1:10- 10 ng/µL) 1 - - - -
StrepDARP. Stock (20 ng/µL) - 0,25 0,25 - -
D2-StrepII Stock 20 ng/µL) - - - 0,25 0,25
Primer iGEM-32 1 1 1 - -
Primer iGEM-33 1 1 1 - -
Flo96 D2_3 fw - - - 1 1
Flo97 D2_3 rv - - - 1 1
Q5-Master mix 25 25 25 25 25
Water 22 22 22 22 22
Total Volume (µl) 50 50 50 50 50

Step Temperature °C Time
1 98 5 min
2 98 30 sec
3 55 30 sec
4 72 60sec
5 Go To 2 31x
6 72 4min
7 8 infinite

The PCR samples were purified with the Omega Gel Extraction Kit according to the enzymatic reaction purification protocol.

18.59 Digestion of amplified of StrepDARPidin for cloning into pET16b

Aim: digest with NcoI and XhoI of the StrepDARPidin PCR product for cloning into the expression vector pET16b & digest of pET16b

The PCR products from 15.58 were cut with NcoI and XhoI to get the restriction sites for cloning into the NcoI/XhoI digested pET16b. This vector was used because of its Amp resistance for fast transformation.

Component PCR StrepDARPidin I (µL) PCR StrepDARPidin II(µL) PCR StrepDARPidin III (µL) Pet16b(µL) Mastermix
StrepDARP. I ( 45 ng/µL) 10 - - - -
StrepDARP. II (70 ng/µL) - 15 - - -
StrepDARP. III (45 ng/µL) - - 10 - -
pET16b ( 58 ng/µL) - - - 17 -
CutSmart 10x - - - - 8
NcoI - - - - 0,5
XhoI - - - - 0,5
Water - - - - 17
Mastermix 10 5 10 - -
Total Volume 20 20 20 20 28

Incubation for 1h at 37°C with heat shock at 85°C for 10 min.

18.60 Ligation of digested StrepDARPidin for cloning into pET16b

Aim: ligation of StrepDARPidin PCR product with the expression vector pET16b

The PCR product was ligated into the NcoI/XhoI digested pET16b. Two attempts were made for a transformation into E. Coli XL1-Blue and BL21.

Component Ligation I (µL) Ligation II (µL) Ligation III (µL) Control
NcoI/XhoI StrepDARPidin (ca. 40  ng/ µL) I/II/III 1 1 1 -
pET16b NcoI/XhoI (45 ng/µL) 5 5 5 5
Ligation Buffer 10x 2 2 2 2
Ligase 1 1 1 1
Water 11 11 11 11
Total Volume 20 20 20 20

Incubation for 1,5 hours at rom temperature with heat shock at 85°C for 10 min. The 20 µL from ligation I were used to transform E. coli XLI-Blue, which was plated out on LB-Amp. Incubation was done overnight at 37°C.

18.61 Gibson assembly of piGEM-005 & D2-StrepII

Aim: Insertion of Hag-D2-Strep into piGEM-005

Component Hag-D2-Strep I(µL) Hag-D2-Strep II (µL)
Gibson-mix 15 15
Insert I
(Hag-D2-Strep I 60 ng/µL)
2,5 -
Insert II
(Hag-D2-Strep II 70 ng/µL)
- 2,5
piGEM-005 SpeI (50 ng/ µL) 2,5 2,5
Total Volume 20 20

Incubation was done  at 50°C for 1 hour with following transformation into E. coli XL1-Blue, plated out on LB-Amp plates and incubated overnight at 37°C.

13.78 colony PCR of colonies of Gibson and ligation (13.76 and 13.77)

Aim: check for correct ligation/Gibson assembly of the oligos with the piGEM002 plasmid

Several clones were picked and transferred onto another plate, the same colony was used to inoculate a PCR reaction to check the insertion of the oligos.

Mix [µl] Master mix for 45 reactions (µl) Const. promoter Cont. + tetO Mod. Lac promoter Ag promoter new Cu promoter new
Template - Small part Small part Small part Small part Small part
Primer iGEM006 45 - - - - -
Primer iGEM061 - 1 - - - -
Primer iGEM063 - - 1 - - -
Primer iGEM059 - - - 1 - -
Primer iGEM057 - - - - 1 -
Primer iGEM055 - - - - - 1
5x HF buffer 180 - - - - -
Phusion DNA polymerase 1:10 45 - - - - -
Water 585 - - - - -
Master Mix - 19 19 19 19 19
Total Volume 855 20 20 20 20 20

Step Temperature °C Time
1 95 10 min
2 98 30 sec
3 55 30 sec
4 72 60 sec
5 Go To 2 34x
6 72 5 min
7 8 infinite

The positive clones (band at ca 1000 bp) were transferred into LB-amp medium and were incubated at 37°C over night for a miniprep. A new colony PCR was carried out with six colonies of the XLIBlue piGEM002+Ag-promoter.

22.4 Repeated amplification of KS module II

Aim: amplify module II for further purification and fusion PCR with flanks of lacA

The PCR for the KSII was repeated with the Phusion DNA-polymerase. A gradient was carried out from 52°C to 62°C.

Content Master mix KSII (µl)
Template: (gBlock KSII) 4,5 0,5
Primer iGEM044 9 1
Primer iGEM045 9 1
HF Buffer 5x 90 10
Phusion DNA-polymerase 9 1
dNTPs 9 1
Water 319,5 35
Total Volume (µl) 450 50

Step Temperature °C Time KS II
1 95 5 min 5 min
2 95 30 sec 20 sec
3 52-62 30 sec 20 sec
4 72 1 min 45 sec 120 sec
5 Go To 2 30x 33x
6 72 4min 4 min
7 8 infinite infinite

The gel showed many unspecific bands of fewer basepairs than expected. The expected band of ca 2000 bp was very weak but got stronger the higher the annealing temperature was.

22.4 repeated fusion-PCR of the Killswitch modules with the flanks

Aim: fuse the modules with the flanks to integrate them into the B. subtilis genome

For this fusion-PCR the flanks of 22.08.14 and the amplified and purified modules I and III from 28.08.14 were used. The module II from 24.08.14 was separated on an agarose gel, cut out and purified as well.

Content KS I (µl) KS II (µl)
Template KSI (ca 5 ng/µl) 1 -
Template KSII (7 ng/µl) - 1
amyE flank I (1.5 ng/µl) 3 -
amyE flank II (ca. 6.5 ng/µl) 1 -
lacA flank I (ca. 5.5 ng/µl) - 1
lacA flank II (7 ng/µl) - 1
Primer piGEM034 1 -
Primer piGEM037 1 -
Primer piGEM040 - 1
Primer pIGEM043 - 1
HF Buffer 5x 10 10
Phusion DNA-polymerase 1 1
dNTPs 1 1
Water 31 31
Total Volume 50 50

Step Temperature °C Time (min/sec) KSI Time (min/sec) KSII
1 95 5 min 5 min
2 95 30 sec 30 sec
3 55 30 sec 30 sec
4 72 2 min 3 min 10 sec
5 Go To 2 34x 34x
6 72 5 min 5 min
7 8 infinite infinite

The fusion PCR was negative, expected bands would be around 1881 bp for KSI and around 3000 for KSII.

30.08.2014

18.62 Gibson assembly of piGEM-005 & D2-StrepII/ Ligation StrepDARPidin-pET16b

The transformation plates were taken out of the incubator and put in the fridge at 4°C.

31.08.2014

18.63 cPCR with clones from Ligation pET16b & StrepDARPidin

Aim: checking the success of the ligation

The grown clones from the ligation from 18.60 were checked via cPCR with primers iGEM-032 and -033. 40 clones from Ligation plates I-III were picked for cPCR and transferred on a plate before.

Content Master mix  for 40 clones (µL) Clones from Ligation I (µL) Clones from Ligation II(µL) Clones from Ligation III(µL)
Template (colony) - 1 1 1
Primer iGEM-32 20 - - -
Primer iGEM-33 20 - - -
Phusion Buffer 5x 160 - - -
Phusion DNA-polymerase 20 - - -
dNTPs 20 - - -
Water 560 - - -
Master mix - 19 19 19
Total Volume 800 20 20 20

Step Temperature °C Time
1 98 10 min
2 98 20 sec
3 55 20 sec
4 72 60 sec
5 Go To 2 33x
6 72 4 min
7 8 infinite

The positive clones show a band at ca. 900 bp and were used for inoculation minipreps so that a test digest could confirm the insertion of StrepDARPidin.

18.64 cPCR with XLI-Blue clones containing piGEM005 Hag-D2-Strep or Hag-D2-Cup

Aim: Checking insertion of Hag-D2-Cup and Hag-D2-Strep into piGEM-005

In order to check the successful integration of D2-Cup and D2-Strep clones were picked for cPCR and transferred on a LB-Amp master plate. As primers Flo96 (D23-fw) and Flo97 (D23-v) were used. 12 clones were screened.

Mix Master-Mix (14x) D2-Strep
Template - Colony
Primer Flo96 D23-fw 7 -
Primer Flo96 D23-rv 7 -
dNTPs 7 -
5x Phusion Buffer 56 -
Phusion DNA-polymerase 7 -
Water 196 -
Mastermix - 20
Total Volume (µl) 280 20

Step Temperature °C Time
1 98 10 min
2 98 30 sec
3 55 30 sec
4 72 40 sec
5 Go To 2 33x
6 72 4 min
7 8 infinite

The clones I1, I3, I6, II2 and II4 were picked for plasmid isolation and sent for sequencing the next day.