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Latest revision as of 02:19, 5 October 2013

Delftibactin. Bringing it all together.

Let's explore the vision to recycle gold from electronic waste using recombinantly expressed delftibactin.

Week 1

We obtained D. acidovorans DSM-39 from the DSMZ and successfully reproduced the paper of Johnsson et al. D. acidovorans is capable to precipitate solid gold from gold chloride solution as purple-black nanoparticles. Already at low concentrations of gold chloride, gold nonaparticles are precipitated increasing with concentration of gold chloride in solution. In our experiments, precipitation on agar plates worked even better than described in the paper.

Week 3

We were able to dissolve gold-containing parts of an old CPU and established a protocol for recovery of gold as soluble gold salts from electronic waste. Moreover, we were able to show specific precipitation of solid gold by D. acidovorans from this "dissolved electronic waste".

Week 15

Using supernatants from the new Delftia acidovorans strain SPH-1, we showed precipitation of gold chloride solution to gold nanoparticles. Furthermore, we melted the purple-black nanoparticles to shiny solid gold.

Week 19

We optimized growth conditions of D. acidovorans and evaluated endogenous background precipitation of metall ions in the possible target E. coli strains E. coli DH10ß and BL21 DE3 following incubation over night on LB and ACM plates. Delftia acidovorans did not exceed E. coli, most probably due to insufficient cultivation time. When grown on LB plates, neither D. acidovorans nor E. coli showed any reactivity.

Week 20

We continued optimized growth conditions of D. acidovorans and testing various E. coli strains for their endogenous capability to precipitate gold in order to identify the strain with the least background to be used as target strain after 2 and 3 days as well as grown at 30°C and room temperature. A cultivation at 30°C for 3 days was identified as optimal. We also started to establish purification of Delftibactin using HP20 resins and successfully verified presence of Delftibactin in the supernatant of D. acidovorans SHP-1. Additionally, we proved precipitation of gold by the purified Delftibactin and detected it by Micro-TOF File:20130911Malditof.pdf. Moreover, we triple-electroporated the final DelRest construct, the final MMCoA plasmid and the first promissing DelH clone into E. coli DH10ß. The Micro-TOF has to be repeated again next week.

Week 21

We repeated last week's Micro-TOF of the first promossing triple-clone, which did not show detectable expression of Delftibactin.

Week 22

Possible E. coli target strains BL21 DE, DH10ß and NEB Turbo were analyzed for their background expression and indcibility. E. coli BL21 DE was identified as best of these three. It was electroporated with DelRest and pIK8.6 and of these, electrocompetent cells were prepared.

Week 23

E. coli BL21 DE + DelRest + pIK8.6 were elctroporated with the DelH clone C5, which harbors a amino acid substitution at the beginning of DelH. Its capability to precipitate gold from solution was analyzed on ACM plates following induction by IPTG. Due to a contamination, the results were inconclusive. The production of Delftibactin was accessed by Micro-TOF. The results were inconclusive, since the ''D. acidovorans'' positive control was negative, the experiment has to be repeated.

Browse gallery

ACM agar plate with D. acidovorans (left) overlaid with 0.2% HAuCl₄ in 0.5% agarose

Sequences of movie showing gold precipitation in D. acidovorans supernatant using gold concentrations ranging from 0 to 2.55 µg/ml

29-04 - 05-05-13

Reproduction of Johnsson et al. Paper

Aim

Reproduction of Johnsson et al. paper: Precipitating gold with D.acidovorans on agar plate, in culture, the medium of the culture and possibly purified delftibactin.

Preparations

Obtain D. acidovorans from DSMZ
Prapare ACM medium as described
Prepare ACM agar plates

Gold Precipitation on ACM Plates

Culture D. acidovorans for 3 days on agar plates at 30°C
Overlay agar plates with 0.2% HAuCl₄ in 0.5 % soft agarose

Result

ACM agar plate with D. acidovorans (left) overlaid with 0.2% HAuCl₄ in 0.5% agarose

Gold precipitation on ACM agar plate worked even better than described in paper

Gold Precipitation in D. acidovorans Supernatants

Culture D. acidovorans for 3 days in ACM plates
Centrifuge bacteria in liquid culture (10 min 6,000 rpm)
Add 0.2% HAuCl₄ to supernatant and medium control

Result

D. acidovorans Supernatant (top) and ACM negative control (bottom) with (left to right) 600µl, 400µl, 200µl, 100µl, 50µl, 0µl 0.2% HAuCl₄. Far right: top: uncentrifuged solution culture with 600µl 0.2% HAuCl₄, bottom: ACM with 600µl HAuCl₄

The supernatant/ medium with gold was centrifuged after 2 h reaction time. left to right: supernatant with 600µl 0.2% HAuCl₄, ACM with 600µl 0.2% HAuCl₄, supernatant with 400µl 0.2% HAuCl₄, ACM with 400µl 0.2% HAuCl₄, supernatant with 200µl 0.2% HAuCl₄, ACM with 200µl 0.2% HAuCl₄

Gold precipitation with D. acidovorans supernatant worked well

Gold Precipitation in D. acidovorans Supernatants at different Gold Chloride Concentrations

Culture D. acidovorans for 3 days in ACM plates
Centrifuge bacteria in liquid culture (10 min 6,000 rpm)
Add HAuCl₄ to supernatant and medium control

Result

Sequences of movie showing gold precipitation in D. acidovorans supernatant using gold concentrations ranging from 0 to 2.55 µg/ml

Gold precipitation with supernatant worked at very low concentration Gold
Gold precipitation with unpurified culture worked as well

Overview
Lab book

Pins removed from an old CPU.

Green solution of dissolved pins.

Gold chloride solution obtained from an old CPU.

Solid gold precipitated by D. acidovorans.

13-05 - 19-05-13

Gold Recovery from Electronic Waste

As test experiment, pins from an old CPU processor chip were dissolved.

Fig.0 Old motherboard from which we will recycle gold.

Step 1: Pins were mechanically removed and collected in a flask.

Fig.1.1 CPU from which pins will be removed.

Fig.1.2 Removed pins.

Step 2: Flask was put onto heating plate and covered in concentrated HCl.

Fig.2 Pins covered in HCl.

Step 3: HNO₃ was carefully added to the pins and heated.

Fig.3 Evolution NO₂ gas.

Step 4: After several minutes of moderate heating, pins dissolved completely. The solution turned green.

Fig.4 Green solution of dissolved pins.

Step 5: The solution was carefully neutralized to pH ~6 in a step-wise manner using first 5 M, later 0.5 M NaOH.
Step 6: This led to precipitation of copper salts, indicated by the milky green coloring.

Fig.6 Milky solution following neutralization.

Step 7: In order to remove the precipitates, the solution was filtered using a buechner funnel and sterile filtrated (0.2 µm).

Fig.7.1 Filtering solution to remove precipitates.

Fig.7.2 Sterile filtering.

Fig.7.3 Filters used for sterile filteration.

Step 8: The final yellow-green gold containing solution was collected in a volumetric flask.

Fig.8Final gold containing solution.

Precipitation of Gold from Electronic Waste

Protocol

In order to evaluate optimial cultivation temperature of D. acidovorans, glycerol stock was streaked on ACM plates and incubated at RT, 30°C and 37°C ON. The next day, plates were covered with 200 µl 0.5% soft agar supplemented with 27 µl "dissolved electronic waste" solution.

Result

D. acidovorans showed intermediate, postponed purple-black coloring upon addition of "dissolved electronic waste" soft agar. Strongest coloring was observed on plates incubated at room temperature and 30°C. Delftibactin expression seems to be significantly decreased in D. acidovorans cultivated at 37°C. Reduced coloring further indicates lower gold ion concentration in the solution recovered compared to th ecommercially availible gold chloride solution [100 g/l]
We could therefore prove capability of D. acidovorans to specifically recover solid gold from dissolved electronic waste.

Fig.9.1 "Dissolved electronic waste" and D. acidovorans.

Fig.9.2 ACM plates prior to addition of gold chloride solution.

Fig.9.3 ACM plates covered with gold chloride solution T= 0:00 h.

Left plate: RT, upper plate: 30°C, lower right plate: 37°C
Fig.9.4 ACM plates covered with gold chloride solution T= 0:30 h.	Fig.9.5 ACM plates covered with gold chloride solution T= 1:00 h.	Fig.9.6 ACM plates covered with gold chloride solution T= 1:30 h.

Overview
Lab book

Sparkling gold appearing in the melting pot.

Final recovered solid gold collected in tube.

Solid gold recovered from nanoparticles in 2 ml tube.

05-08 - 11-08-13

Melting Gold Nanoparticles to Solid Gold

Step 1: Cultivation of D. acidovorans
200 ml ACM medium were inocculated with recently obtained D. acidovorans SPH-1 from glycerol stock and incubated at 30°C for 3 days.

Step 2: Precipitation of Gold Nanoparticles

D. acidovorans culture in ACM was divided in 50 ml aliquots and centrifuges at 3,750 rpm for 30 min.
Supernatant was transferred to fresh tube.
300 µl gold chloride stock solution [µg/ml] was added to D. acidovorans supernatant, suspention mixed and incubated at RT for 20 min.

Fig.2.1 Supernatant of D. acidovorans.

Fig.2.2 Precipitated gold nanoparticles in D. acidovorans supernatant.

Step 3: Aggregation of gold nanoparticles.

Fig.3 Gold nanoparticle solution on melting pot.

Step 4: Melting gold nanoparticles to solid gold.

Fig.4.1 Heating aggregated gold nanoparticles in melting pot.	Fig.4.2 Heating aggregated gold nanoparticles in melting pot upside down.	Fig.4.3 Remaining solid gold in melting pot.	Fig.4.3 Sparkling gold appearing in the melting pot.
Fig.4.4 Final recovered solid gold collected in tube.

Alternatively, we also performed the protocol described above in a 2 ml tube.

Fig.5 Solid gold recovered from nanoparticles in 2 ml tube.

Lab book

02-09 - 08-09-13

Optimizing Growth Conditions of D.acidovorans and Evaluation of Endogenous Background Gold Precipitation

In order to optimize plates for cultivation of D.acidovorans as well as to evaluate of endogenous capability of possible target strains, capability of D.acidovorans and E. coli DH10ß and BL21 DE3 grown on LB and ACM plates was tested.

On Agar Plate following ON Incubation at 30°C

On agar plates, D.acidovorans, E.coli DH10ß, and Bl21 were plated from liquid culture and grown ON at 30 °C.
The following day, 1.6 ml soft agar (0.5%) was mixed with goldchloride [100 g/l] as follows:

Final goldchloride concentration [mM]	Volume goldchloride stock [µl]
5	27
10	54.4
20	108.8

Agar plate	Goldchloride [mM]	H₂O	D.acidovorans	DH10ß	BL21 DE3
ACM	0	no reaction	no reaction	no reaction	no reaction
ACM	5	postponed weak coloring ON	weak coloring after 2 min	weak coloring after 2 min	weak coloring after 2 min
ACM	10	postponed dark coloring ON	strong coloring after 2 min	strong coloring after 2 min	strong coloring after 2 min
ACM	20	postponed dark coloring ON	strong coloring after 2 min	strong coloring after 2 min	strong coloring after 2 min
LB	0	no reaction	no reaction	no reaction	no reaction
LB	5	no reaction	no reaction	no reaction	no reaction
LB	10	no reaction	no reaction	no reaction	no reaction
LB	20	no reaction	no reaction	no reaction	no reaction

Result

Evaluation of Bacteria regarding Background Gold Precipitation on Agar Plate following ON Incubation at 30°C
ACM plate before incubation with gold chloride containing soft agar.	ACM plate after incubation with gold chloride containing soft agar counter-clockwise beginning from top left in concentrations of 0 mM, 5 mM, 10 mM, 20 mM.

All bacteria showed strong and immediate purple-black coloring indicating gold precipitation depending on gold chloride concentration. Regarding the precipitation of gold, there does not seem to be a significant advantage of D.acidivorans. In contrast to the paper from Johnston et al., we only grew them ON at 30°C rather than for 3 days. We will repeat the experiment using 3 day incubation time and also evaluate gold precipitation on additional media as well as by additional bacteria.

Overview
Lab book

Test if purified Delftibactin is able to precipitate gold. From left to right: ACM media, filtered supernatant D. acidovorans, purified Delftibactin

Test if purified Delftibactin (diluted 1:10 in H₂O) is able to precipitate gold. From left to right: water, 1:10 ACM media, 1:10 supernatant D. acidovorans, 1:10 filtered supernatant D. acidovorans, 1:10 purified Delftibactin

09-09 - 15-09-13

Optimizing Growth Conditions of D.acidovorans and Evaluation of Endogenous Background Gold Precipitation

In order to optimize plates, growth time and incubation temperature of D.acidovorans as well as to evaluate of endogenous capability of possible target strains, various growth conditions and bacteria were tested.

On Agar Plate following 3 Day Incubation at 30°C

Media was chelex treated, agar added and autoclaved, before plates were poured.
On agar plates, D.acidovorans, E.coli DH10ß, Bl21 DE3, and BL21 pLys as well as MG1655 and Bacillus subtilus were plated from liquid culture and grown for three days at 30 °C.
14.4 ml soft agar (0.5%) was mixed with gold chloride [100 g/l] as follows and 200 µl per quarter plate added:

Final gold chloride concentration [mM]	Volume goldchloride stock [µl]
0	0
0.5	2.7
2.5	13.5
5	27

Result

Agar plate	Goldchloride [mM]	H₂O	D.acidovorans	BL21 DE3	BL21 pLys	H₂O	DH10ß	B.circulans	MG1655
ACM	0	no reaction	no reaction	no reaction	no reaction	no reaction	no reaction	no reaction	no reaction
ACM	0.5	no reaction	no reaction	no reaction	no reaction	no reaction	no reaction	no reaction	no reaction
ACM	2.5	minor coloring	minor coloring	no reaction	no reaction	no reaction	no reaction	minor coloring	no reaction
ACM	5	strong coloring	strong coloring	intermediate coloring	minor coloring	minor coloring	minor coloring	minor coloring	minor coloring
LB	0	no reaction	no reaction	no reaction	no reaction	no reaction	no reaction	no reaction	no reaction
LB	0.5	no reaction	no reaction	no reaction	no reaction	no reaction	no reaction	no reaction	no reaction
LB	2.5	no reaction	no reaction	no reaction	no reaction	no reaction	no reaction	no reaction	no reaction
LB	5	no reaction	no reaction	no reaction	no reaction	no reaction	no reaction	no reaction	no reaction
M9	0	no reaction	no reaction	no reaction	no reaction	no reaction	no reaction	no reaction	no reaction
M9	0.5	minor coloring	no reaction	no reaction	no reaction	no reaction	no reaction	no reaction	no reaction
M9	2.5	strong coloring	minor coloring	no reaction	no reaction	strong coloring	minor coloring	strong coloring	no reaction
M9	5	strong coloring	minor coloring	no reaction	no reaction	minor coloring	minor coloring	minor coloring	no reaction

Gold precipitation on ACM plates incubated for 3 days at 30°C
Fig.1.1 Gold precipitation on ACM plates incubated for 3 days at 30°C: prior to addition of soft agar.	Fig.1.2 Gold precipitation on ACM plates incubated for 3 days at 30°C: T= 0:00.	Fig.1.3 Gold precipitation on ACM plates incubated for 3 days at 30°C: T= 0:20.	Fig.1.4 Gold precipitation on ACM plates incubated for 3 days at 30°C: T= 0:50.
Fig.1.5 Gold precipitation on ACM plates incubated for 3 days at 30°C: T= 1:00.	Fig.1.6 Gold precipitation on ACM plates incubated for 3 days at 30°C: T= 2:00.	Fig.1.7 Gold precipitation on ACM plates incubated for 3 days at 30°C: T= 3:00.

Gold precipitation on LB plates incubated for 3 days at 30°C
Fig.2.1 Gold precipitation on LB plates incubated for 3 days at 30°C: T= 0:00.	Fig.2.2 Gold precipitation on LB plates incubated for 3 days at 30°C: T= 0:10.	Fig.2.3 Gold precipitation on LB plates incubated for 3 days at 30°C: T= 0:20.	Fig.2.4 Gold precipitation on LB plates incubated for 3 days at 30°C: T= 0:50.
Fig.2.5 Gold precipitation on LB plates incubated for 3 days at 30°C: T= 2:00.

Gold precipitation on M9 plates incubated for 3 days at 30°C
Fig.3.1 Gold precipitation on M9 plates incubated for 3 days at 30°C: T= 0:00.	Fig.3.2 Gold precipitation on M9 plates incubated for 3 days at 30°C: T= 0:10.	Fig.3.3 Gold precipitation on M9 plates incubated for 3 days at 30°C: T= 0:20.	Fig.3.4 Gold precipitation on M9 plates incubated for 3 days at 30°C: T= 0:50.
Fig.3.5 Gold precipitation on M9 plates incubated for 3 days at 30°C: T= 2:00.

Unfortunately, the streaked E.coli DH10ß were contaminated (obvious only on LB plates) and can threfore not be analyzed.
On ACM plates following 30°C incubation, D.acidovorans, B.circulans, E.coli BL21 DE3 as well as the negative control next to D.acidovorans, but not the one on plate B, showed immediate purple-black coloring indicating formation of gold nanoparticles at 5 mM gold chloride. At 2.5 mM, D.acidovorans and the neighbouring negative control as well as B.circulans showed some coloring. E.coli BL21 pLys and MG1655 only showed minor coloring at 5 mM. At lower concentrations, no gold precipitation was observed.

=> E.coli BL21 pLys and MG1655 are promissing candidates for expression since they show the least background activity.

=> Optimal gold chloride concentration lies between 2.5 and 5 mM.

=> Delftibactin can diffuse through agar.

On LB plates following 30°C incubation, none of the tested bacteria showed any reaction.

=> LB is not suitable for expression of NRPs.

=> What is missing in LB that does not allow metall precipitating activity?

On M9 plates following 30°C incubation (which were mistakenly autoclaved after addition of glucose), D.acidovorans hardly grew. At 5 mM and 2.5 mM gold chloride, there is heavy background activity on the negative controls. Additionally, B.circulans shows strong - D.acidovorans minor formation of nanoparticles.

=> M9 is not suitable for gold precipitation experiments.

On Agar Plate following 3 Day Incubation at RT

Media was chelex treated, agar added and autoclaved, before plates were poured.
On agar plates, D.acidovorans, E.coli DH10ß, Bl21 DE3, and BL21 pLys as well as MG1655 and Bacillus subtilus were plated from liquid culture and grown for three days at RT.
14.4 ml soft agar (0.5%) was mixed with gold chloride [100 g/l] as follows and 200 µl per quarter plate added:

Final gold chloride concentration [mM]	Volume goldchloride stock [µl]
0	0
0.5	2.7
2.5	13.5
5	27

Result

Agar plate	Goldchloride [mM]	H₂O	D.acidovorans	BL21 DE3	BL21 pLys	H₂O	DH10ß	B.circulans	MG1655
ACM	0	no reaction	no reaction	no reaction	no reaction	no reaction	no reaction	no reaction	no reaction
ACM	0.5	no reaction	no reaction	no reaction	no reaction	no reaction	no reaction	no reaction	no reaction
ACM	2.5	no reaction	no reaction	no reaction	no reaction	minor coloring	minor coloring	intermediate coloring	minor coloring
ACM	5	strong coloring	strong coloring	minor coloring	minor coloring	intermediate coloring	strong coloring	strong coloring	strong coloring
LB	5	no reaction	no reaction	no reaction	no reaction	no reaction	no reaction	no reaction	no reaction
M9	5	strong coloring	strong coloring	no reaction	no reaction	strong coloring	strong coloring	strong coloring	no reaction

Gold precipitation on ACM plates incubated for 3 days at RT
Fig.4.1 Gold precipitation on ACM plates incubated for 3 days at RT: T= 0:00.	Fig.4.2 Gold precipitation on ACM plates incubated for 3 days at RT: T= 0:10.	Fig.4.3 Gold precipitation on ACM plates incubated for 3 days at RT: T= 0:20.	Fig.4.4 Gold precipitation on ACM plates incubated for 3 days at RT: T= 1:00.

Gold precipitation on LB plates incubated for 3 days at RT
Fig.5.1 Gold precipitation on LB plates incubated for 3 days at RT: T= 0:00.	Fig.5.2 Gold precipitation on LB plates incubated for 3 days at RT: T= 0:10.	Fig.5.3 Gold precipitation on LB plates incubated for 3 days at RT: T= 0:20.	Fig.5.4 Gold precipitation on LB plates incubated for 3 days at RT: T= 1:00.

Gold precipitation on M9 plates incubated for 3 days at RT
Fig.6.1 Gold precipitation on M9 plates incubated for 3 days at RT: T= 0:00.	Fig.6.2 Gold precipitation on M9 plates incubated for 3 days at RT: T= 0:10.	Fig.6.3 Gold precipitation on M9 plates incubated for 3 days at RT: T= 0:20.	Fig.6.4 Gold precipitation on M9 plates incubated for 3 days at RT: T= 1:00.

On ACM plates following RT incubation, D.acidovorans and the neighbouring negative control, B.circulans and E.coli MG1655 show minor immediate coloring at 5 mM. At lower concentrations, no activity can be observed.

=> There is less Delftibactin expression in D.acidovorans at lower temperatures.

=> E.coli BL21 pLys remains a promissing candidate for expression since they show the least background activity.

On LB plates following RT incubation, there is again nothing to see.

On M9 plates following RT incubation, there is again high background at negative controls as well as intermediate coloring for D.acidovorans and B.circulans.
We will therefore analyze gold precipitation capacities of supernatants of D.acidovorans, E.coli DH10ß, BL21 DE3, BL21 pLys and MG1655 in ACM liquid medium following 48 and 72 h.

In ACM Medium following 2 Day Incubation at 30°C

In 5 ml ACM medium, D.acidovorans, E.coli DH10ß, Bl21 DE3, and BL21 pLys as well as MG1655 and pure medium were inocculated and grown for two days at 30°C.
1 ml supernatant was mixed with gold chloride [100 g/l] as follows:

Final gold chloride concentration [µg/ml]	Volume goldchloride stock [µl]
0.6	6

Result

Following addition of gold chloride solution, E.coli DH10ß, Bl21 DE3, and BL21 pLys showed rapid purple-black coloring indicating gold nanoparticle formation. They exceed the capacity of D.acidovorans, which showed gold precipitation slightly faster than E.coli MG1655. The ACM did not show any background activity.
We will extend the incubation time of the cultures further to evaluate differences, possibly due to accumulation of Delftibactin in the medium.

Evaluation of Bacteria regarding Background Gold Precipitation in ACM liquid Medium following 2 day Incubation at 30°C
Fig.1.1 From left: 1 ml ACM medium, supernatants of E.coli DH10ß, MG1655, Bl21 pLys and BL21 DE3 as well as D.acidovorans and 6 µl gold chloride stock solution in lid.	Fig.1.2 From left: 1 ml ACM medium, supernatants of E.coli DH10ß, MG1655, Bl21 pLys and BL21 DE3 as well as D.acidovorans prior to mixing.	Fig.1.3 From left: 1 ml ACM medium, supernatants of E.coli DH10ß, MG1655, Bl21 pLys and BL21 DE3 as well as D.acidovorans: T= 0:00 s. Particles on to bottom are undissolved gold chloride.	Fig.1.4 From left: 1 ml ACM medium, supernatants of E.coli DH10ß, MG1655, Bl21 pLys and BL21 DE3 as well as D.acidovorans: T= 30 s.
Fig.1.5 From left: 1 ml ACM medium, supernatants of E.coli DH10ß, MG1655, Bl21 pLys and BL21 DE3 as well as D.acidovorans: T= 1:00 min.	Fig.1.6 From left: 1 ml ACM medium, supernatants of E.coli DH10ß, MG1655, Bl21 pLys and BL21 DE3 as well as D.acidovorans: T= 2:00 min.	Fig.1.7 From left: 1 ml ACM medium, supernatants of E.coli DH10ß, MG1655, Bl21 pLys and BL21 DE3 as well as D.acidovorans: T= 3:00 min.	Fig.1.8 From left: 1 ml ACM medium, supernatants of E.coli DH10ß, MG1655, Bl21 pLys and BL21 DE3 as well as D.acidovorans: T= 4:00 min.
Fig.1.9 From left: 1 ml ACM medium, supernatants of E.coli DH10ß, MG1655, Bl21 pLys and BL21 DE3 as well as D.acidovorans: T= 5:00 min.

In ACM Medium following 3 day Incubation at 30°C

In 5 ml ACM medium, D.acidovorans, E.coli DH10ß, Bl21 DE3, and BL21 pLys as well as MG1655 were inocculated and grown for three days at 30°C.
1 ml supernatant was mixed with gold chloride [100 g/l] as follows:

Final gold chloride concentration [µg/ml]	Volume goldchloride stock [µl]
0.6	6

Result

Again, E. coli DH10ß, Bl21 DE3, and BL21 pLys showed rapid purple-black coloring exceeding the capacity of D.acidovorans and E. coli MG1655. These results are highly inconclusive and the experiment has to be repeated.

Evaluation of Bacteria regarding Background Gold Precipitation in ACM liquid Medium following 3 day Incubation at 30°C
Fig.1.1 From left: 1 ml ACM medium, supernatants of E.coli DH10ß, MG1655, Bl21 pLys and BL21 DE3 as well as D.acidovorans and 6 µl gold chloride stock solution in lid.	Fig.1.2 From left: 1 ml ACM medium, supernatants of E.coli DH10ß, MG1655, Bl21 pLys and BL21 DE3 as well as D.acidovorans prior to mixing.	Fig.1.3 From left: 1 ml ACM medium, supernatants of E.coli DH10ß, MG1655, Bl21 pLys and BL21 DE3 as well as D.acidovorans: T= 0:00 min. Particles on to bottom are undissolved gold chloride.	Fig.1.4 From left: 1 ml ACM medium, supernatants of E.coli DH10ß, MG1655, Bl21 pLys and BL21 DE3 as well as D.acidovorans: T= 1:00 min.
Fig.1.5 From left: 1 ml ACM medium, supernatants of E.coli DH10ß, MG1655, Bl21 pLys and BL21 DE3 as well as D.acidovorans: T= 2:00 min.	Fig.1.6 From left: 1 ml ACM medium, supernatants of E.coli DH10ß, MG1655, Bl21 pLys and BL21 DE3 as well as D.acidovorans: T= 5:00 min.	Fig.1.7 From left: 1 ml ACM medium, supernatants of E.coli DH10ß, MG1655, Bl21 pLys and BL21 DE3 as well as D.acidovorans: T= 6:00 min.	Fig.1.8 From left: 1 ml ACM medium, supernatants of E.coli DH10ß, MG1655, Bl21 pLys and BL21 DE3 as well as D.acidovorans: T= 7:00 min.
Fig.1.9 From left: 1 ml ACM medium, supernatants of E.coli DH10ß, MG1655, Bl21 pLys and BL21 DE3 as well as D.acidovorans: T= 10:00 min.	Fig.1.10 From left: 1 ml ACM medium, supernatants of E.coli DH10ß, MG1655, Bl21 pLys and BL21 DE3 as well as D.acidovorans: T= 13:00 min.	Fig.1.11 From left: 1 ml ACM medium, supernatants of E.coli DH10ß, MG1655, Bl21 pLys and BL21 DE3 as well as D.acidovorans: T= 15:00 min.

Purification of Delftibactin

Purification

Grew 1 L of D. acidovorans SPH1 for 24h in ACM media at 30°C. (2x 500 mL ACM 15 mL about 3 days old D. acidovorans culture in ACM)
Centrifugation of culture at 3750 rpm for 30 min
Retrieved supernatant
Added 20 g/L HP-20
Stirred at RT for 2 h
Filtration with Buchner funnel to retain HP-20
Washed with 400 ml dddH₂O
Elution with 400 ml Methanol
Evaporated methanol with rotary evaporator
Resuspended in 2 ml 50 methanol : 50 ddH₂O

Testing Purified Delftibactin

Different intermediate products were tested for their ability to precipitate gold. For this, 3 µl of AuCl₃ (100g/l) were added to 500 µl solution. The outcome is presented in the following table.

Sample	Precipitating Gold?	Colour of Precipitate	Time until Precipitation Started
AMC media	yes	grey	~2 h
Supernatant of D. acidovorans SPH-1 culture	yes	black	1:30 min
Supernatant after treatment with HP20	yes	black	1:40 min
HP20 after elution step	no	--	--
Methanol	no	--	--
Methanol used for elution	no	--	--

Sample	Diluted in Water	Volume sample	Gold Solution 100g/l	Precipitating gold?	Colour of Precipitate	Time until Precipitation Started
ACM media	--	160 µl	1 µl	yes	black	1 h
Supernatant of D. acidovorans in ACM, filtered	--	160 µl	1 µl	yes	black	1 h
Purified Delftibactin after dilution in 50:50 Methanol, Water	--	160 µl	1 µl	no	--	--
Water	--	250 µl	1.5 µl	no	--	--
ACM media	1:10	250 µl	1.5 µl	no	--	--
Supernatant of D. acidovorans in ACM	1:10	250 µl	1.5 µl	no	--	--
Supernatant of D. acidovorans in ACM, filtered	1:10	250 µl	1.5 µl	no	--	--
Purified Delftibactin after dilution in 50:50 Methanol, H₂O	1:10	250 µl	1.5 µl	yes	gray	over night

Testing Purified Delftibactin for Capability to Precipitate Gold
Test if purified Delftibactin is able to precipitate gold. From left to right: ACM media, filtered supernatant D. acidovorans, purified Delftibactin	Test if purified Delftibactin (diluted 1:10 in H₂O) is able to precipitate gold. From left to right: water, 1:10 ACM media, 1:10 supernatant D. acidovorans, 1:10 filtered supernatant D. acidovorans, 1:10 purified Delftibactin

Micro-TOF

The supernatant of a 24 h D. acidovorans SPH-1 culture was analysed before and after the purification procedure using a MICRO-TOF. As negative control unpurified ACM media was measured as well.
File:Heidelberg 20130911Malditof.pdf

Result

Delftibactin was clearly present in the purified supernatant of the D. acidovorans SPH-1 culture. In contrast it could not be detected in the ACM media or the supernatant which was not purified.

Triple Clone pIK8.6, DelH and DelH I 6B

Preparations

Electrocompetent E. coli DH10ß and BL21 DE3 were prepared as described.

Electroporation

We electroporated all three plasmids into E. coli DH10ß using pIK8.6. Although the DelH clone I 6B has a deletion in the ribosome binding site, we hope that we still get enough expression of DelH.

Electroporation	Total amount of DNA [ng]	Ratio pIK2.6: DelH:DelRest	pIK8.6-1(12 Kb, 146 ng/µl)	DelH-I6B midi (23 Kb, 235.2 ng/µl)	DelRest (32 Kb, 2.4 µg/µl)
Triple into E. coli DH10ß	774.2	1:2:3	1 µl of 1:10 (146 ng)	1 µl (235.2 ng)	1.5 µl of 1:10 (375 ng)

Preparation of Cultures for Purification and MICRO-TOF

Bacteria	Media	Antibiotics
--	ACM	--
E. coli DH10ß	ACM	--
Delftia acidovorans SPH-1	ACM	--
E. coli DH10ß with plasmids pHM04 (I6B), pFSN (D8w) & pIK8.6	ACM	Ampicillin, Chloramphenicol, Kanamycin

Inoculation of 10 ml of over night cultures of each sample
Growth at 30°C (Delftia acidovorans) and 37°C (E. coli DH10ß and E. coli DH10ß + plasmids pHM04 (I6B), pFSN (D8w) & pIK8.6)
For each culture, 500 ml of ACM media with antibiotics was inoculated with the pre-cultures
The cultures were grown for about 8 h.

ODs

E. coli DH10ß = 1.2
Delftia acidovorans SPH-1 = 0.4
E. coli DH10ß + plasmids pHM04 (I6B), pFSN (D8w) & pIK8.6 = 0.8

Purification of Delftibactin

Centrifugation of cultures at 3750 rpm for 30 min
Retrieved supernatant
Added 20 g/L HP-20
Stirred at RT for 2 h
Filtration with Buchner funnel to retain HP-20
Washed with 400 ml dddH₂O
Elution with 200 ml Methanol
Evaporated methanol with rotary evaporator
Resuspended in 2 ml 50 methanol : 50 ddH₂O

MICRO-TOF

File:Heidelberg Microtof0913.pdf

Result

Apparently the purification of delftibactin did not work, as even in the positive control (D. acidovorans SPH-1) no delftibactin was detected. As the cultures were harvested after only a short time of growth it is suspected that maybe the production of delftibactin had not started yet. The next time the cultures should grow for longer.

Lab book

17-09 - 22-09-13

Triple Clone pIK2.6, DelH and DelH I 6B

Electroporation

We electroporated all three plasmids again into E. coli BL21 DE3. This time, we incubated them them for 3 full days.

Electroporation	Total amount of DNA [ng]	Ratio pIK8.6: DelH:DelRest	pIK8.6-1(12 Kb)	DelH-I6B midi (23 Kb, 235.2 ng/µl)	DelRest (32 Kb, 2.4 µg/µl)
Triple into E. coli DH10ß	774.2	1:2:3	1 µl of 1:10 (146 ng)	1 µl (235.2 ng)	1.5 µl of 1:10 (375 ng)

Preparation of Cultures for Purification and MICRO-TOF

Bacteria	Media	Antibiotics
--	ACM	--
Delftia acidovorans SPH-1	ACM	Ampicillin
E. coli BL21 DE3	ACM	--
E. coli BL21 DE3 with plasmids pHM04 (I6B), pFSN (D8w) & pIK8.6	ACM	Ampicillin, Chloramphenicol, Kanamycin

Inoculation of 10 ml of over night cultures of each sample
For DH10β with plasmids pHM04 (I6B), pFSN (D8w) & pIK8.6 no such culture was made
Growth at 30°C
For each culture 1 L of ACM media with antibiotics was inoculated with this pre-culture
1 L of ACM media with antibiotics was directly inoculated with 1 mL of a 3 hour culture DH10β with plasmids pHM04 (I6B), pFSN (D8w) & pIK8.6 which had originally been made from a clone which had been picked from the agar plate. The 1 L culture was started 3 hours later than the other cultures
Expression was induced in E. coli BL21 DE3 with 1 mM IPTG after 8 h of growth
Cultures were harvested after 64 hours of growth

Purification of Delftibactin

Centrifugation of cultures at 3750 rpm for 30 min
Retrieved supernatant
Added 20 g/L HP-20
Stirred at RT for 2 h
Filtration with Buchner funnel to retain HP-20
Washed with 400 ml dddH₂O
Elution with 400 ml Methanol
Evaporated methanol with rotary evaporator
Resuspended in 2 ml 50 methanol : 50 ddH₂O

MICRO-TOF

File:Heidelberg Microtof0925.pdf

Result

Only in the culture of D. acidovorans SPH-1 Delftibactin could be detected. The samples were negative for delftibactin. Apparently our triple clone does not produce delftibactin. However, this is not surprising, since the DelH clone I 6B harbors a mutation in the promoter region.

Overview
Lab book

mRPF Expression in E. coli BL21 DE3, DH10ß and NEB Turbo 16 h post iduction.

mRPF Expression in E. coli BL21 DE3, DH10ß and NEB Turbo 16 h post iduction in liquid media.

23-09 - 29-09-13

Evaluation of Background Expression and Indcibility of Possible Target Strains

Electroporation of mRFP Controls

1 µl miniprpep DNA of the backbones pSB6A1 and pSB3C5 containing mRFP were elctroporated in E.coli Bl21 DE3. Cells were streaked on LB Amp + Chlor plates and stored at RT for two days.

Result

All plates were heavily overgrown. As discussed next, the obtained BL21 DE3 were actually BL21 DE3 pLys and therefore resitant to chloramphenicol. Electroporation has to be repeated, after real E. coli BL21 DE3 are obtained.

New BL21 DE3

We obtained new BL21 DE3 from the Russel Lab, which were obtained from NEB directly. Cells were inocculated in 10 ml LB and streaked on LB Chlor plates. The vial was frozen again at -80°C.

Electroporation of mRFP Controls

Real E. coli BL21 DE3, DH10ß and NEB Turbo were co-electroporated with 1 µl miniprpep DNA of the backbones pSB6A1 and pSB3C5 containing mRFP to check for leaky ecpression. Transformed bacteria were streaked on 2YT plates with Amp + Chlor and incubated ON at 30°C.
The next day, expression of mRFP on backbones pSB6A1 and pSB3C5 was induced by 50 µl 1 mM IPTG as well as inocculated in YT Amp + Chlor + 1 mM IPTG.

Result

After 6 h, only BL21 DE showed expression of mRFP. Yet, it was not restricted to the induced area. Another 10 h later, all strains showed red colonies with and without induction. E. coli BL21 DE3 showed least background expression and intermediate expression upon induction on plate and in liquid media.

mRPF Expression in BL21 DE3, DH10ß and NEB Turbo with and without Induction
Fig.1.1 mRPF Expression in E. coli BL21 DE3 6 h post iduction.	Fig.1.2 mRPF Expression in E. coli BL21 DE3 6 h post iduction.	Fig.1.3 mRPF Expression in E. coli DH10ß 6 h post iduction.	Fig.1.4 mRPF Expression in E. coli NEB Turbo 6 h post iduction.
Fig.1.5 mRPF Expression in E. coli NEB Turbo 6 h post iduction.	Fig.1.6 mRPF Expression in E. coli BL21 DE3, DH10ß and NEB Turbo 16 h post iduction.	Fig.1.7 mRPF Expression in E. coli BL21 DE3, DH10ß and NEB Turbo 16 h post iduction in liquid media.

Electrocompetent E. coli BL21 DE + DelRest and pIK8.6

Electroporation of DelRest, DelH and pIK8.6

As stated above, BL21 DE3 showed least background expression of mRFP and were therefore chosen for preparation of electrocompetent cells as described. Cells were streaked on 2YT Kan + Chlor plates and incubated at RT for 2 days.

Colony-PCR Conditions CP.W22.A

6 colonies were analyzed for Del Rest and pIK8.6 by colony PCR.

DelL

Reagent	Amount [µl]
Colonies	-
FS_14 10 µM	1
FS_15 10 µM	1
OneTaq Master Mix	10
DMSO	1
ddH₂O	7

Cycles	Temperature [°C]	Time [s]
1	95	300
12	95	30
	66 ↓ 0.5	30
	72	90
18	95	30
	63	30
	72	90
1	72	720
1	12	inf

pIK8.6

Reagent	Amount [µl]
Colonies	-
VF2 10 µM	1
IK_25 10 µM	1
OneTaq Master Mix	10
DMSO	1
ddH₂O	7

Cycles	Temperature [°C]	Time [s]
1	95	300
35	95	30
	54	30
	72	90
1	72	600
1	12	inf

Result

Expected band: ~1.4 Kb (DelL) and ~1 Kb (pIK8.6)

Fig22.2 Screening of DelRest + pIK8.6 Co-transformants

All of the analyzed colonies shows expected band for DelL.

=> Inocculate 2 ml 2YT Kan + Chlor with 2 colonies for preparation of electrocompetent cells

Electrocompetent Cells

Electrocompetent E. coli BL21 DE3 + DelRest + pIK8.6 wer prepared as described.

Lab book

30-09 - 06-10-13

Triple Clone pIK.6, DelRest and DelH C5

Electroporation

E. coli BL21 DE3 + DelRest + pIK8.6 were electroporated with DelH C5 miniprep DNA and streaked on 2YT + Amp + Chlor + Kan plates and incubated at RT for 2 days.

Evaluation of Gold Precipitation on ACM Plates

In order to evaluate gold nanoparticle formation capacity DelH clone C5 in presence of DelRest and pIK8.6, they were streaked on ACM plates as follows and incubated at 30°C for 3 days. As controls, we used the BL21 DE3 transformed with pSB3C5 and pSB6A1.

Plate	Antibiotic	Cells
ACM	-	D.acidovorans
		E.coli BL21 DE3 + pSB3C5 + pSB6A1
		E.coli BL21 DE3 + DelH C5 + DelRest + pIK8.6
		empty
ACM	Amp + Chlor + Kan	D.acidovorans
		E.coli BL21 DE3 + pSB3C5 + pSB6A1
		E.coli BL21 DE3 + DelH C5 + DelRest + pIK8.6
		empty
ACM + IPTG	Amp + Chlor + Kan	D.acidovorans
		E.coli BL21 DE3 + pSB3C5 + pSB6A1
		E.coli BL21 DE3 + DelH C5 + DelRest + pIK8.6
		empty

Following 3 day incubation, 200 µl 5 mM gold chloride in soft agar was applied.

Result

Evaluation of Gold Precipitation of C5 Triple Clone on ACM Plates following 3 day Incubation at 30°C
Fig.1.1 From left: background control, E.coli BL21 DE3 containing ampicillin and chloramphenicol parts, E.coli BL21 DE3 containing DelH C5, DelRest and pIK8.6 and D.acidovorans prior to addition of gold chloride soft agar.	Fig.1.2 rom left: background control, E.coli BL21 DE3 containing ampicillin and chloramphenicol parts, E.coli BL21 DE3 containing DelH C5, DelRest and pIK8.6 and D.acidovorans: T= 0:00.	Fig.1.3 rom left: background control, E.coli BL21 DE3 containing ampicillin and chloramphenicol parts, E.coli BL21 DE3 containing DelH C5, DelRest and pIK8.6 and D.acidovorans: T= 0:15.	Fig.1.4 rom left: background control, E.coli BL21 DE3 containing ampicillin and chloramphenicol parts, E.coli BL21 DE3 containing DelH C5, DelRest and pIK8.6 and D.acidovorans: T= 0:30.
Fig.1.5 rom left: background control, E.coli BL21 DE3 containing ampicillin and chloramphenicol parts, E.coli BL21 DE3 containing DelH C5, DelRest and pIK8.6 and D.acidovorans: T= 1:00.	Fig.1.6 rom left: background control, E.coli BL21 DE3 containing ampicillin and chloramphenicol parts, E.coli BL21 DE3 containing DelH C5, DelRest and pIK8.6 and D.acidovorans: T= 1:30.

Unexpectedly, the D.acidovorans control grew on the ACM + Amp + Chlor + Kan plates, indicating a contamination with triple resistant bacteria. Therefore, the experiment is inconclusive and has to be repeated. Moreover, the background control shows significant activity.
Nevertheless, there was neither increased activity of the induced 'E.coli BL21 DE3 containing DelH C5, DelRest and pIK8.6 compared to the E.coli BL21 DE3 negative control nor to the not induced control.

Preparation of Cultures for Purification and MICRO-TOF

Bacteria	Media	Antibiotics
E. coli BL21 DE3	ACM	--
Delftia acidovorans SPH-1	ACM	--
E. coli BL21 DE3 with plasmids pHM04 (C5), pFSN (D8w) & pIK8.6	ACM	Ampicillin, Chloramphenicol, Kanamycin

Inoculation of 10 ml of over night cultures of each sample
Growth at 30°C
For each culture, 1 L of ACM media with antibiotics was inoculated with the pre-culture
Expression was induced in all E. coli BL21 DE3 with 1 mM IPTG after 24 h of growth
All cultures were supplemented with 10 mM sodium propionate after 24 h of growth
The cultures selected with ampicillin were supplemented with an additional 1:2000 ampicillin after 24 h of growth
Cultures were harvested after 48 hours of growth and 24 hours after induction

Purification of Delftibactin

Centrifugation of cultures at 3750 rpm for 45 min

Protocol for liquid culture

Retrieved supernatant
Added 20 g/L HP-20
Stirred at RT for 2 h
Filtration with Buchner funnel to retain HP-20
Washed with 400 ml dddH₂O
Elution with 400 ml Methanol
Evaporated methanol with rotary evaporator
Resuspended in 2 ml 50 methanol : 50 ddH₂O

Protocol for pellet

Kept pellet
Washed pellet with destilled water
Resuspended pellet in 50 ml destilled water
Freeze and thawed 8 times
Centrifugation at 3750 rpm for 30 min
Kept supernatant
Added 20 g/L HP-20
Stirred at RT for 2 h
Filtration with Buchner funnel to retain HP-20
Washed with 400 ml dddH₂O
Elution with 400 ml Methanol
Evaporated methanol with rotary evaporator
Resuspended in 2 ml 50 methanol : 50 ddH₂O

MICRO-TOF

File:Heidelberg 20131004 MicroTOF.pdf

Result

Unexpectedly, the D. acidovorans positive control was negative for expression of Delftibactin. The results are therefore inconclusive and the experiment has to be repeated.

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    <div class="container">
              <!--Project Description-->
-             <div style="margin-top:80px">
+             <div style="margin-top:10%;">
-                         <h1><span style="font-size:180%;color:#FFCC00;">Delftibactin.</span><span class="text-muted" style="font-family:Arial, sans-serif; font-size:120%"> Bringing it all together.</span></h1>
+                         <h1><span style="font-size:180%;color:#FFCC00;">Delftibactin.</span><span class="text-muted" style="font-size:120%"> Bringing it all together.</span></h1>
-                         <p>Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet.</p>
+                         <p style="font-size:14px">Let's explore the vision to recycle gold from electronic waste using recombinantly expressed delftibactin.</p>
              </div>
              <div class="row">
-             <div class="col-sm-4">
+             <div class="col-sm-6">
              <!--Months-->
-                 <ul class="pagination" style="margin-bottom:2%; margin-left:4%;">
+                 <ul class="pagination" style="margin-bottom:2%; margin-left:15%;">
-                    <!--<button type="button" class="btn btn-default">May</button>
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-                    <button type="button" class="btn btn-default">October</button>-->
                    <li><a href="#" id="backwards">&laquo;</a></li>
-                   <li class="active month_tab" id="may"><a href="#" style="width:90px; text-align:center">May</a></li>
+                   <li class="active month_tab" id="may"><a href="#" style="width:100px; text-align:center">May</a></li>
-                   <li class="month_tab" id="june"><a href="#" style="width:90px; text-align:center">June</a></li>
+                   <!--<li class="month_tab" id="june"><a href="#" style="width:100px; text-align:center">June</a></li>
-                   <li class="month_tab" id="july"><a href="#" style="width:90px; text-align:center" >July</a></li>
+                   <li class="month_tab" id="july"><a href="#" style="width:100px; text-align:center" >July</a></li>-->
-                   <li class="month_tab" id="august"><a href="#" style="width:90px; text-align:center">August</a></li>
+                   <li class="month_tab" id="august"><a href="#" style="width:100px; text-align:center">August</a></li>
-                   <li class="month_tab" id="september"><a href="#" style="width:90px; text-align:center">September</a></li>
+                   <li class="month_tab" id="september"><a href="#" style="width:100px; text-align:center">September</a></li>
-                   <!--<li class="month_tab" id="october"><a href="#" style="width:90px; text-align:center">October</a></li>-->
+                   <!--<li class="month_tab" id="october"><a href="#" style="width:100px; text-align:center">October</a></li>-->
                    <li><a href="#" id="forwards">&raquo;</a></li>
                  </ul>
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                      <div id="myCarousel" class="carousel slide">
                          <!-- Indicators -->
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                          <div class="carousel-inner">
-                           <div class="item active may first">
+                            <div class="item active may first">
                                  <div class="container">
                                      <div class="carousel-caption scrollContent2" data-spy="scroll" data-target="#navbarExample" data-offset="0" id="week1">
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                                        <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">
-This week the project DelRest was launched.
+We obtained <i>D. acidovorans</i> DSM-39 from the DSMZ and successfully reproduced the paper of Johnsson <i>et al. D. acidovorans</i> is capable to precipitate solid gold from gold chloride solution as purple-black nanoparticles. Already at low concentrations of gold chloride, gold nonaparticles are precipitated increasing with concentration of gold chloride in solution. In our experiments, precipitation on agar plates worked even better than described in the paper.
-In order to clone the Delftibactin cluster from <i>D. Acidovorans </i> into <i> E.coli </i>  we decided to use Gibson cloning. Therefore Gibson primers were designed to amplify our target backbone pSB4K5 with an overlap to DelA. Furthermore the Gibson primer connecting DelOP and DelAG introduces the ribosome binding site BBa_BNILS. Accordingly the Gibson primer for joining DelL with DelOP includes the ribosome binding site BBa_BNILS. The very last gibson primer used to amplify DelL consequently has an overlap to the beginning of the mRFP reporter gene connected to pSB4K5. Check out our vectormap if you are curious about the detailed primer design. The goal of this week was to amplify our previously assembled backbone with the intended overlaps as well as the desired genes from our host organism <i>D. Acidovorans </i>.
                                         </p>
@@ Line 76: / Line 64: @@
                                  </div>
                              </div>
-                            <div class="item may">
-                                <div class="container">
-                                    <div class="carousel-caption scrollContent2" data-spy="scroll" data-target="#navbarExample" data-offset="0" id="week1">
-                                      <h1>Week 2</h1>
-                                      <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">
-This week the project DelRest was launched.
-In order to clone the Delftibactin cluster from <i>D. Acidovorans </i> into <i> E.coli </i>  we decided to use Gibson cloning. Therefore Gibson primers were designed to amplify our target backbone pSB4K5 with an overlap to DelA. Furthermore the Gibson primer connecting DelOP and DelAG introduces the ribosome binding site BBa_BNILS. Accordingly the Gibson primer for joining DelL with DelOP includes the ribosome binding site BBa_BNILS. The very last gibson primer used to amplify DelL consequently has an overlap to the beginning of the mRFP reporter gene connected to pSB4K5. Check out our vectormap if you are curious about the detailed primer design. The goal of this week was to amplify our previously assembled backbone with the intended overlaps as well as the desired genes from our host organism <i>D. Acidovorans </i>.
-                                       </p>
-                                    </div>
-                                </div>
-                            </div>
-                             <div class="item may">
-                                <div class="container">
-                                    <div class="carousel-caption scrollContent2" data-spy="scroll" data-target="#navbarExample" data-offset="0" id="week1">
-                                      <h1>Week 3</h1>
-                                      <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">
-This week the project DelRest was launched.
-In order to clone the Delftibactin cluster from <i>D. Acidovorans </i> into <i> E.coli </i>  we decided to use Gibson cloning. Therefore Gibson primers were designed to amplify our target backbone pSB4K5 with an overlap to DelA. Furthermore the Gibson primer connecting DelOP and DelAG introduces the ribosome binding site BBa_BNILS. Accordingly the Gibson primer for joining DelL with DelOP includes the ribosome binding site BBa_BNILS. The very last gibson primer used to amplify DelL consequently has an overlap to the beginning of the mRFP reporter gene connected to pSB4K5. Check out our vectormap if you are curious about the detailed primer design. The goal of this week was to amplify our previously assembled backbone with the intended overlaps as well as the desired genes from our host organism <i>D. Acidovorans </i>.
-                                       </p>
-                                    </div>
-                                </div>
-                            </div>
-                          <div class="item may">
-                                <div class="container">
-                                    <div class="carousel-caption scrollContent2" data-spy="scroll" data-target="#navbarExample" data-offset="0" id="week1">
-                                      <h1>Week 4</h1>
-                                      <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">
-This week the project DelRest was launched.
-In order to clone the Delftibactin cluster from <i>D. Acidovorans </i> into <i> E.coli </i>  we decided to use Gibson cloning. Therefore Gibson primers were designed to amplify our target backbone pSB4K5 with an overlap to DelA. Furthermore the Gibson primer connecting DelOP and DelAG introduces the ribosome binding site BBa_BNILS. Accordingly the Gibson primer for joining DelL with DelOP includes the ribosome binding site BBa_BNILS. The very last gibson primer used to amplify DelL consequently has an overlap to the beginning of the mRFP reporter gene connected to pSB4K5. Check out our vectormap if you are curious about the detailed primer design. The goal of this week was to amplify our previously assembled backbone with the intended overlaps as well as the desired genes from our host organism <i>D. Acidovorans </i>.
-                                       </p>
-                                    </div>
-                                </div>
-                            </div>
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-                                    <div class="carousel-caption scrollContent2" data-spy="scroll" data-target="#navbarExample" data-offset="0" id="week1">
-                                      <h1>Week 5</h1>
-                                      <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">
-This week the project DelRest was launched.
-In order to clone the Delftibactin cluster from <i>D. Acidovorans </i> into <i> E.coli </i>  we decided to use Gibson cloning. Therefore Gibson primers were designed to amplify our target backbone pSB4K5 with an overlap to DelA. Furthermore the Gibson primer connecting DelOP and DelAG introduces the ribosome binding site BBa_BNILS. Accordingly the Gibson primer for joining DelL with DelOP includes the ribosome binding site BBa_BNILS. The very last gibson primer used to amplify DelL consequently has an overlap to the beginning of the mRFP reporter gene connected to pSB4K5. Check out our vectormap if you are curious about the detailed primer design. The goal of this week was to amplify our previously assembled backbone with the intended overlaps as well as the desired genes from our host organism <i>D. Acidovorans </i>.
-                                       </p>
-                                    </div>
-                                </div>
-                            </div>
-                           <div class="item june first">
-                                <div class="container">
-                                    <div class="carousel-caption scrollContent2" data-spy="scroll" data-target="#navbarExample" data-offset="0" id="week1">
-                                      <h1>Week 6</h1>
-                                      <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">
-This week the project DelRest was launched.
-In order to clone the Delftibactin cluster from <i>D. Acidovorans </i> into <i> E.coli </i>  we decided to use Gibson cloning. Therefore Gibson primers were designed to amplify our target backbone pSB4K5 with an overlap to DelA. Furthermore the Gibson primer connecting DelOP and DelAG introduces the ribosome binding site BBa_BNILS. Accordingly the Gibson primer for joining DelL with DelOP includes the ribosome binding site BBa_BNILS. The very last gibson primer used to amplify DelL consequently has an overlap to the beginning of the mRFP reporter gene connected to pSB4K5. Check out our vectormap if you are curious about the detailed primer design. The goal of this week was to amplify our previously assembled backbone with the intended overlaps as well as the desired genes from our host organism <i>D. Acidovorans </i>.
-                                       </p>
-                                    </div>
-                                </div>
-                            </div>
-                             <div class="item june">
-                                <div class="container">
-                                    <div class="carousel-caption scrollContent2" data-spy="scroll" data-target="#navbarExample" data-offset="0" id="week1">
-                                      <h1>Week 7</h1>
-                                      <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">
-This week the project DelRest was launched.
-In order to clone the Delftibactin cluster from <i>D. Acidovorans </i> into <i> E.coli </i>  we decided to use Gibson cloning. Therefore Gibson primers were designed to amplify our target backbone pSB4K5 with an overlap to DelA. Furthermore the Gibson primer connecting DelOP and DelAG introduces the ribosome binding site BBa_BNILS. Accordingly the Gibson primer for joining DelL with DelOP includes the ribosome binding site BBa_BNILS. The very last gibson primer used to amplify DelL consequently has an overlap to the beginning of the mRFP reporter gene connected to pSB4K5. Check out our vectormap if you are curious about the detailed primer design. The goal of this week was to amplify our previously assembled backbone with the intended overlaps as well as the desired genes from our host organism <i>D. Acidovorans </i>.
-                                       </p>
-                                    </div>
-                                </div>
-                            </div>
-                           <div class="item june">
-                                <div class="container">
-                                    <div class="carousel-caption scrollContent2" data-spy="scroll" data-target="#navbarExample" data-offset="0" id="week1">
-                                      <h1>Week 8</h1>
-                                      <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">
-This week the project DelRest was launched.
-In order to clone the Delftibactin cluster from <i>D. Acidovorans </i> into <i> E.coli </i>  we decided to use Gibson cloning. Therefore Gibson primers were designed to amplify our target backbone pSB4K5 with an overlap to DelA. Furthermore the Gibson primer connecting DelOP and DelAG introduces the ribosome binding site BBa_BNILS. Accordingly the Gibson primer for joining DelL with DelOP includes the ribosome binding site BBa_BNILS. The very last gibson primer used to amplify DelL consequently has an overlap to the beginning of the mRFP reporter gene connected to pSB4K5. Check out our vectormap if you are curious about the detailed primer design. The goal of this week was to amplify our previously assembled backbone with the intended overlaps as well as the desired genes from our host organism <i>D. Acidovorans </i>.
-                                       </p>
-                                    </div>
-                                </div>
-                            </div>
-                            <div class="item june last">
-                                <div class="container">
-                                    <div class="carousel-caption scrollContent2" data-spy="scroll" data-target="#navbarExample" data-offset="0" id="week1">
-                                      <h1>Week 9</h1>
-                                      <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">
-This week the project DelRest was launched.
-In order to clone the Delftibactin cluster from <i>D. Acidovorans </i> into <i> E.coli </i>  we decided to use Gibson cloning. Therefore Gibson primers were designed to amplify our target backbone pSB4K5 with an overlap to DelA. Furthermore the Gibson primer connecting DelOP and DelAG introduces the ribosome binding site BBa_BNILS. Accordingly the Gibson primer for joining DelL with DelOP includes the ribosome binding site BBa_BNILS. The very last gibson primer used to amplify DelL consequently has an overlap to the beginning of the mRFP reporter gene connected to pSB4K5. Check out our vectormap if you are curious about the detailed primer design. The goal of this week was to amplify our previously assembled backbone with the intended overlaps as well as the desired genes from our host organism <i>D. Acidovorans </i>.
-                                       </p>
-                                    </div>
-                                </div>
-                            </div>
-                            <div class="item july first">
-                                <div class="container">
-                                    <div class="carousel-caption scrollContent2" data-spy="scroll" data-target="#navbarExample" data-offset="0" id="week1">
-                                      <h1>Week 10</h1>
-                                      <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">
-This week the project DelRest was launched.
-In order to clone the Delftibactin cluster from <i>D. Acidovorans </i> into <i> E.coli </i>  we decided to use Gibson cloning. Therefore Gibson primers were designed to amplify our target backbone pSB4K5 with an overlap to DelA. Furthermore the Gibson primer connecting DelOP and DelAG introduces the ribosome binding site BBa_BNILS. Accordingly the Gibson primer for joining DelL with DelOP includes the ribosome binding site BBa_BNILS. The very last gibson primer used to amplify DelL consequently has an overlap to the beginning of the mRFP reporter gene connected to pSB4K5. Check out our vectormap if you are curious about the detailed primer design. The goal of this week was to amplify our previously assembled backbone with the intended overlaps as well as the desired genes from our host organism <i>D. Acidovorans </i>.
-                                       </p>
-                                    </div>
-                                </div>
-                            </div>
-                            <div class="item july">
                                <div class="container">
                                  <div class="carousel-caption scrollContent2" data-spy="scroll" data-target="#navbarExample" data-offset="0">
-                                   <h1>Week 11</h1>
+                                   <h1>Week 3</h1>
-                                   <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">
+                                   <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">We were able to dissolve gold-containing parts of an old CPU and established a protocol for recovery of gold as soluble gold salts from electronic waste. Moreover, we were able to show specific precipitation of solid gold by <i>D. acidovorans</i> from this "dissolved electronic waste".
-Cras justo odio, dapibus ac facilisis in, egestas eget quam. Donec id elit non mi porta gravida at eget metus. Nullam id dolor id nibh ultricies vehicula ut id elit.</p>
-                                  <p><a class="btn btn-large btn-primary" href="#">Learn more</a></p>
-                                  <p>At the beginning of the week, we could verify that the Gibson Assembly for Tripeptide I was indeed positive, however, the other Gibson Assemblies did not work properly. Instead of picking new colonies, we decided to optimize the Gibson recipe instead, as backbone religations were the most common problem. With these improved protocols, we used Gibson Assembly for the Dipeptide, Tripeptide II and Tetrapeptide I, later that week, Tetrapeptide II followed. After the Transformation to DH10β cells and screening by restriction digest we could send samples for the Dipeptide and Tetrapeptide I to sequencing and obtained a positive alignment. Hence we transformed BAP I cells with the positive constructs. The same was...
                                    </p>
                                  </div>
                                </div>
                              </div>
-                            <div class="item july">
-                              <div class="container">
-                                <div class="carousel-caption scrollContent2" data-spy="scroll" data-target="#navbarExample" data-offset="0">
-                                  <h1>Week 12</h1>
-                                  <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">
-Cras justo odio, dapibus ac facilisis in, egestas eget quam. Donec id elit non mi porta gravida at eget metus. Nullam id dolor id nibh ultricies vehicula ut id elit.</p>
-                                  <p><a class="btn btn-large btn-primary" href="#">Browse gallery</a></p>
-                                  <p>At the beginning of the week, we could verify that the Gibson Assembly for Tripeptide I was indeed positive, however, the other Gibson Assemblies did not work properly. Instead of picking new colonies, we decided to optimize the Gibson recipe instead, as backbone religations were the most common problem. With these improved protocols, we used Gibson Assembly for the Dipeptide, Tripeptide II and Tetrapeptide I, later that week, Tetrapeptide II followed. After the Transformation to DH10β cells and screening by restriction digest we could send samples for the Dipeptide and Tetrapeptide I to sequencing and obtained a positive alignment. Hence we transformed BAP I cells with the positive constructs. The same was...
-                                  </p>
-                                </div>
-                              </div>
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-                            <div class="item july">
-                              <div class="container">
-                                <div class="carousel-caption scrollContent2" data-spy="scroll" data-target="#navbarExample" data-offset="0">
-                                  <h1>Week 13</h1>
-                                  <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">Cras justo odio, dapibus ac facilisis in, egestas eget quam. Donec id elit non mi porta gravida at eget metus. Nullam id dolor id nibh ultricies vehicula ut id elit.</p>
-                                  <p><a class="btn btn-large btn-primary" href="#">Browse gallery</a></p>
-                                </div>
-                              </div>
-                            </div>
-                            <div class="item july last">
-                              <div class="container">
-                                <div class="carousel-caption scrollContent2" data-spy="scroll" data-target="#navbarExample" data-offset="0">
-                                  <h1>Week 14</h1>
-                                  <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">At the beginning of the week, we could verify that the Gibson Assembly for Tripeptide I was indeed positive, however, the other Gibson Assemblies did not work properly. Instead of picking new colonies, we decided to optimize the Gibson recipe instead, as backbone religations were the most common problem. With these improved protocols, we used Gibson Assembly for the Dipeptide, Tripeptide II and Tetrapeptide I, later that week, Tetrapeptide II followed.</p>
-                                </div>
-                              </div>
-                            </div>
                              <div class="item august first">
-                              <img src="data:image/png;base64,"/>
                                <div class="container">
                                  <div class="carousel-caption scrollContent2" data-spy="scroll" data-target="#navbarExample" data-offset="0">
                                    <h1>Week 15</h1>
-                                   <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">Cras justo odio, dapibus ac facilisis in, egestas eget quam. Donec id elit non mi porta gravida at eget metus. Nullam id dolor id nibh ultricies vehicula ut id elit.</p>
+                                   <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">Using supernatants from the new <i>Delftia acidovorans</i> strain SPH-1, we showed precipitation of gold chloride solution to gold nanoparticles. Furthermore, we melted the purple-black nanoparticles to shiny solid gold.  </p>
-                                  <p><a class="btn btn-large btn-primary" href="#">Learn more</a></p>
                                  </div>
                                </div>
                              </div>
-                             <div class="item august">
+                             <div class="item august last">
-                              <div class="container">
-                                <div class="carousel-caption scrollContent2" data-spy="scroll" data-target="#navbarExample" data-offset="0">
-                                  <h1>Week 16</h1>
-                                  <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">Cras justo odio, dapibus ac facilisis in, egestas eget quam. Donec id elit non mi porta gravida at eget metus. Nullam id dolor id nibh ultricies vehicula ut id elit.</p>
-                                  <p><a class="btn btn-large btn-primary" href="#">Browse gallery</a></p>
-                                </div>
-                              </div>
-                            </div>
-                            <div class="item august">
-                              <img src="data:image/png;base64,"/>
-                              <div class="container">
-                                <div class="carousel-caption scrollContent2" data-spy="scroll" data-target="#navbarExample" data-offset="0">
-                                  <h1>Week 17</h1>
-                                  <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">Cras justo odio, dapibus ac facilisis in, egestas eget quam. Donec id elit non mi porta gravida at eget metus. Nullam id dolor id nibh ultricies vehicula ut id elit.</p>
-                                  <p><a class="btn btn-large btn-primary" href="#">Browse gallery</a></p>
-                                </div>
-                              </div>
-                            </div>
-                            <div class="item august">
-                              <img src="data:image/png;base64,"/>
-                              <div class="container">
-                                <div class="carousel-caption scrollContent2" data-spy="scroll" data-target="#navbarExample" data-offset="0">
-                                  <h1>Week 18</h1>
-                                  <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">dumm dummy </p>
-                                </div>
-                              </div>
-                            </div>
-                           <div class="item august last">
                                <img src="data:image/png;base64,"/>
                                <div class="container">
                                  <div class="carousel-caption scrollContent2" data-spy="scroll" data-target="#navbarExample" data-offset="0">
                                    <h1>Week 19</h1>
-                                   <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">After successful dumm dummy </p>
+                                   <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">We optimized growth conditions of D. acidovorans and evaluated endogenous background precipitation of metall ions in the possible target E. coli strains E. coli DH10ß and BL21 DE3 following incubation over night on LB and ACM plates. <i>Delftia acidovorans</i> did not exceed <i>E. coli</i>, most probably due to insufficient cultivation time. When grown on LB plates, neither <i>D. acidovorans</i> nor <i>E. coli</i> showed any reactivity. </p>
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                                    <h1>Week 20</h1>
-                                   <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">dumm dummy </p>
+                                   <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">We continued optimized growth conditions of <i>D. acidovorans</i> and testing various <i>E. coli</i> strains for their endogenous capability to precipitate gold in order to identify the strain with the least background to be used as target strain after 2 and 3 days as well as grown at 30°C and room temperature. A cultivation at 30°C for 3 days was identified as optimal. We also started to establish purification of Delftibactin using HP20 resins and successfully verified presence of Delftibactin in the supernatant of <i>D. acidovorans</i> SHP-1. Additionally, we proved precipitation of gold by the purified Delftibactin and detected it by Micro-TOF File:20130911Malditof.pdf. Moreover, we triple-electroporated the final DelRest construct, the final MMCoA plasmid and the first promissing DelH clone into <i>E. coli</i> DH10ß. The Micro-TOF has to be repeated again next week. </p>
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                                    <h1>Week 21</h1>
-                                   <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">dummy </p>
+                                   <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">We repeated last week's Micro-TOF of the first promossing triple-clone, which did not show detectable expression of Delftibactin. </p>
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                                    <h1>Week 22</h1>
-                                   <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">dummmmmy</p>
+                                   <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">Possible E. coli target strains BL21 DE, DH10ß and NEB Turbo were analyzed for their background expression and indcibility. E. coli BL21 DE was identified as best of these three. It was electroporated with DelRest and pIK8.6 and of these, electrocompetent cells were prepared. </p>
                                  </div>
                                </div>
@@ Line 336: / Line 125: @@
                                  <div class="carousel-caption scrollContent2" data-spy="scroll" data-target="#navbarExample" data-offset="0">
                                    <h1>Week 23</h1>
-                                   <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">Cras justo odio, dapibus ac facilisis in, egestas eget quam. Donec id elit non mi porta gravida at eget metus. Nullam id dolor id nibh ultricies vehicula ut id elit.</p>
+                                   <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">E. coli BL21 DE + DelRest + pIK8.6 were elctroporated with the DelH clone C5, which harbors a amino acid substitution at the beginning of DelH. Its capability to precipitate gold from solution was analyzed on ACM plates following induction by IPTG. Due to a contamination, the results were inconclusive. The production of Delftibactin was accessed by Micro-TOF. The results were inconclusive, since the ''D. acidovorans'' positive control was negative, the experiment has to be repeated. </p>
                                    <p><a class="btn btn-large btn-primary" href="#">Browse gallery</a></p>
                                  </div>
                                </div>
                              </div>
                      </div>
                      <a class="left carousel-control" href="#myCarousel" data-slide="prev"><span class="glyphicon glyphicon-chevron-left"></span></a>
@@ Line 348: / Line 135: @@
                  </div>
              </div><!-- /.carousel -->
+         </div>
+        <div class="col-sm-6">
+          <div class="methods jumbotron" data-spy="scroll" data-target="#navbarExample" data-offset="0" style="height:364px">
+                <h3>Graphical Abstract</h3>
+                <div style="height:200px;">
+<a class="fancybox fancyGraphical" rel="group" href="https://static.igem.org/mediawiki/2013/e/e3/Heidelberg_ga_delf.png">
+    <img style="height:200px; margin-bottom:10px; padding:1%;border-style:solid;border-width:1px;border-radius: 4px; border-color: grey;" src="https://static.igem.org/mediawiki/2013/e/e3/Heidelberg_ga_delf.png"></img>
-            <div class="jumbotron methods" data-spy="scroll" data-target="#navbarExample" data-offset="0">
+</a>
-                <h2>Methods:</h2>
-                <p style="font-size:10pt; text-align:justify">Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet.Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. .
-                </p>
              </div>
          </div>
-        <div class="col-sm-8">
+      </div>
-            <!--Start Weekly Labjournal-->
+     <div class="row">
-             <!--Week 10-->
+             <!--Start Weekly Labjournal-->
+            <!--Week1-->
              <div class="labjournal-weekly">
                  <div>
                      <ul class="nav nav-tabs">
-                       <li class="active"><a href="#a10" data-toggle="tab">Overview</a></li>
+                       <li class="active"><a href="#a1" data-toggle="tab">Overview</a></li>
-                       <li><a href="#b10" data-toggle="tab">DelA-E</a></li>
+                       <li><a href="#b1" data-toggle="tab">Lab book</a></li>
-                      <li><a href="#c10" data-toggle="tab">DelA-F</a></li>
-                      <li><a href="#d10" data-toggle="tab">DelA-G</a></li>
-                      <li><a href="#e10" data-toggle="tab">DelE-G</a></li>
-                      <li><a href="#j10" data-toggle="tab">DelF-G</a></li>
-                      <li><a href="#f10" data-toggle="tab">DelG</a></li>
-                      <li><a href="#g10" data-toggle="tab">DelO-P</a></li>
-                      <li><a href="#h10" data-toggle="tab">DelL</a></li>
-                      <li><a href="#i10" data-toggle="tab">pSB4K5</a></li>
                      </ul>
                  </div>
                  <div class="jumbotron">
-                     <div class="scrollContent nav navbar" data-spy="scroll" data-target="#navbarExample" data-offset="0">
+                     <div class="nav navbar" data-spy="scroll" data-target="#navbarExample" data-offset="0">
                          <div class="tab-content">
-                             <div class="tab-pane active" id="a10">
+                             <div class="tab-pane active" id="a1">
+                                  </html>{{:Team:Heidelberg/Templates/Delftibactin_overview1}}<html>
                                  <p style="font-size:12pt; text-align:justify;">
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="b10">
-                                <p style="font-size:12pt; text-align:justify;">
-                                     </html>{{:Team:Heidelberg/Templates/Del_week10_AE}}<html>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="c10">
-                                <p style="font-size:12pt; text-align:justify;">
-                                </html>{{:Team:Heidelberg/Templates/Del_week10_AF}}<html>
                                  </p>
                              </div>
-                             <div class="tab-pane" id="d10">
+                             <div class="tab-pane" id="b1">
                                  <p style="font-size:12pt; text-align:justify;">
+                                 </html>{{:Team:Heidelberg/Templates/Delftibactin_week1}}<html>
-                                 </html>{{:Team:Heidelberg/Templates/Del_week10_AG}}<html>
                                  </p>
                              </div>
-                             <div class="tab-pane" id="e10">
-                                <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week10_EG}}<html>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="j10">
-                                <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week10_FG}}<html>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="f10">
-                                <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week10_G}}<html>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="g10">
-                                <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week10_OP}}<html>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="h10">
-                                <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week10_L}}<html>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="i10">
-                                <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week10_pSB4K5}}<html>
-                                </p>
-                            </div>
                          </div>
                      </div>
@@ Line 444: / Line 184: @@
              </div>
-            <!--Week 11-->
+             <!--Week3-->
              <div class="labjournal-weekly">
                  <div>
                      <ul class="nav nav-tabs">
-                       <li class="active"><a href="#a11" data-toggle="tab">Overview</a></li>
+                       <li class="active"><a href="#a3" data-toggle="tab">Overview</a></li>
-                       <li><a href="#b11" data-toggle="tab">DelA-E</a></li>
+                       <li><a href="#b3" data-toggle="tab">Lab book</a></li>
-                      <li><a href="#d11" data-toggle="tab">DelA-G</a></li>
-                      <li><a href="#e11" data-toggle="tab">DelE-G</a></li>
-                      <li><a href="#c11" data-toggle="tab">DelF-G</a></li>
-                      <li><a href="#f11" data-toggle="tab">DelG</a></li>
-                      <li><a href="#g11" data-toggle="tab">DelO-P</a></li>
-                      <li><a href="#j11" data-toggle="tab">DelL-P</a></li>
-                      <li><a href="#h11" data-toggle="tab">DelL</a></li>
-                      <li><a href="#i11" data-toggle="tab">pSB4K5</a></li>
                      </ul>
                  </div>
-                 <div class="jumbotron weekly">
+                 <div class="jumbotron">
-                     <div class="scrollContent nav navbar" data-spy="scroll" data-target="#navbarExample" data-offset="0">
+                     <div class="nav navbar" data-spy="scroll" data-target="#navbarExample" data-offset="0">
-                       <div class="tab-content">
-                            <div class="tab-pane active" id="a11">
-                                <p style="font-size:12pt; text-align:justify;">
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="b11">
-                                <p style="font-size:12pt; text-align:justify;">
-                                     </html>{{:Team:Heidelberg/Templates/Del_week11_AE}}<html>
-                                </p>
-                            </div>
-                             <div class="tab-pane" id="d11">
+                        <div class="tab-content">
+                             <div class="tab-pane active" id="a3">
                                  <p style="font-size:12pt; text-align:justify;">
+                                  </html>{{:Team:Heidelberg/Templates/Delftibactin_overview3}}<html>
-                                </html>{{:Team:Heidelberg/Templates/Del_week11_AG}}<html>
                                  </p>
                              </div>
-                             <div class="tab-pane" id="e11">
+                             <div class="tab-pane" id="b3">
                                  <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week11_EG}}<html>
+                                       </html>{{:Team:Heidelberg/Templates/Delftibactin_week3}}<html>
                                  </p>
                              </div>
-                             <div class="tab-pane" id="f11">
+                        </div>
-                                <p style="font-size:12pt; text-align:justify;">
+                    </div>
-                               </html>{{:Team:Heidelberg/Templates/Del_week11_G}}<html>
+                </div>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="j11">
-                                <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week11_LP}}<html>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="g11">
-                                <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week11_OP}}<html>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="h11">
-                                <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week11_L}}<html>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="i11">
-                                <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week11_pSB4K5}}<html>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="c11">
-                                <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week11_FG}}<html>
-                                </p>
-                            </div>
-                       </div>
-                   </div>
-               </div>
              </div>
-                    <!--Week 12-->
+            <!--Week15-->
              <div class="labjournal-weekly">
                  <div>
                      <ul class="nav nav-tabs">
-                       <li class="active"><a href="#a12" data-toggle="tab">Overview</a></li>
+                       <li class="active"><li><a href="#a15" data-toggle="tab">Overview</a></li>
-                      <li><a href="#b12" data-toggle="tab">DelA-E</a></li>
+                       <li><a href="#b15" data-toggle="tab">Lab book</a></li>
-                       <li><a href="#e12" data-toggle="tab">DelE-G</a></li>
-                      <li><a href="#c12" data-toggle="tab">DelF-G</a></li>
-                      <li><a href="#f12" data-toggle="tab">DelG</a></li>
-                      <li><a href="#h12" data-toggle="tab">DelL-P</a></li>
-                      <li><a href="#g12" data-toggle="tab">DelO-P</a></li>
                      </ul>
                  </div>
-                    <div class="jumbotron weekly">
+                <div class="jumbotron">
-                     <div class="scrollContent nav navbar" data-spy="scroll" data-target="#navbarExample" data-offset="0">
+                     <div class="nav navbar" data-spy="scroll" data-target="#navbarExample" data-offset="0">
                          <div class="tab-content">
-                             <div class="tab-pane active" id="a12">
+                             <div class="tab-pane active" id="a15">
                                  <p style="font-size:12pt; text-align:justify;">
+                                  </html>{{:Team:Heidelberg/Templates/Delftibactin_overview15}}<html>
                                  </p>
                              </div>
-                             <div class="tab-pane" id="b12">
+                             <div class="tab-pane" id="b15">
                                  <p style="font-size:12pt; text-align:justify;">
-                                     </html>{{:Team:Heidelberg/Templates/Del_week12_AE}}<html>
+                                       </html>{{:Team:Heidelberg/Templates/Delftibactin_week15}}<html>
                                  </p>
                              </div>
-                            <div class="tab-pane" id="e12">
-                                <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week12_EG}}<html>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="f12">
-                                <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week12_G}}<html>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="h12">
-                                <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week12_LP}}<html>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="g12">
-                                <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week12_OP}}<html>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="c12">
-                                <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week12_FG}}<html>
-                                </p>
-                            </div>
                          </div>
                      </div>
@@ Line 599: / Line 247: @@
              </div>
-                 <!--Week 13-->
+            <!--Week19-->
              <div class="labjournal-weekly">
                  <div>
                      <ul class="nav nav-tabs">
-                       <li class="active"><a href="#a13" data-toggle="tab">Overview</a></li>
+                       <li class="active"><a href="#b19" data-toggle="tab">Lab book</a></li>
-                      <li><a href="#b13" data-toggle="tab">DelA-E</a></li>
-                      <li><a href="#c13" data-toggle="tab">DelA-F</a></li>
-                      <li><a href="#d13" data-toggle="tab">DelA-G</a></li>
-                      <li><a href="#h13" data-toggle="tab">DelE</a></li>
-                      <li><a href="#e13" data-toggle="tab">DelE-G</a></li>
-                      <li><a href="#j13" data-toggle="tab">DelF-G</a></li>
-                      <li><a href="#f13" data-toggle="tab">DelG</a></li>
-                      <li><a href="#g13" data-toggle="tab">DelO-P</a></li>
                      </ul>
                  </div>
-                 <div class="jumbotron weekly">
+                 <div class="jumbotron">
-                     <div class="scrollContent nav navbar" data-spy="scroll" data-target="#navbarExample" data-offset="0">
+                     <div class="nav navbar" data-spy="scroll" data-target="#navbarExample" data-offset="0">
-                         <div class="tab-content">
-                            <div class="tab-pane active" id="a13">
-                                <p style="font-size:12pt; text-align:justify;">
-                                </p>
-                            </div>
-                             <div class="tab-pane" id="b13">
+                             <div class="tab-pane" id="b19">
                                  <p style="font-size:12pt; text-align:justify;">
-                                     </html>{{:Team:Heidelberg/Templates/Del_week13_AE}}<html>
+                                       </html>{{:Team:Heidelberg/Templates/Delftibactin_week19}}<html>
                                  </p>
                              </div>
-                             <div class="tab-pane" id="c13">
-                                <p style="font-size:12pt; text-align:justify;">
-                                </html>{{:Team:Heidelberg/Templates/Del_week13_AF}}<html>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="d13">
-                                <p style="font-size:12pt; text-align:justify;">
-                                </html>{{:Team:Heidelberg/Templates/Del_week13_AG}}<html>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="h13">
-                                <p style="font-size:12pt; text-align:justify;">
-                                </html>{{:Team:Heidelberg/Templates/Del_week13_E}}<html>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="e13">
-                                <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week13_EG}}<html>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="f13">
-                                <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week13_G}}<html>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="g13">
-                                <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week13_OP}}<html>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="j13">
-                                <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week13_FG}}<html>
-                                </p>
-                            </div>
                          </div>
                      </div>
                  </div>
-            </div>
-             <!--Week 14-->
+            <!--Week20-->
              <div class="labjournal-weekly">
                  <div>
                      <ul class="nav nav-tabs">
-                       <li class="active"><a href="#a14" data-toggle="tab">Overview</a></li>
+                       <li class="active"><a href="#a20" data-toggle="tab">Overview</a></li>
-                       <li><a href="#b14" data-toggle="tab">DelA-E</a></li>
+                       <li><a href="#b20" data-toggle="tab">Lab book</a></li>
-                      <li><a href="#c14" data-toggle="tab">DelA-F</a></li>
-                      <li><a href="#d14" data-toggle="tab">DelA-G</a></li>
-                      <li><a href="#j14" data-toggle="tab">DelF-G</a></li>
-                      <li><a href="#f14" data-toggle="tab">DelG</a></li>
-                      <li><a href="#g14" data-toggle="tab">DelO-P</a></li>
                      </ul>
                  </div>
-                 <div class="jumbotron weekly">
+                 <div class="jumbotron">
-                     <div class="scrollContent nav navbar" data-spy="scroll" data-target="#navbarExample" data-offset="0">
+                     <div class="nav navbar" data-spy="scroll" data-target="#navbarExample" data-offset="0">
-                         <div class="tab-content">
-                             <div class="tab-pane active" id="a14">
+                        <div class="tab-content">
+                             <div class="tab-pane active" id="a20">
                                  <p style="font-size:12pt; text-align:justify;">
+                                  </html>{{:Team:Heidelberg/Templates/Delftibactin_overview20}}<html>
                                  </p>
                              </div>
-                             <div class="tab-pane" id="b14">
+                             <div class="tab-pane" id="b20">
                                  <p style="font-size:12pt; text-align:justify;">
-                                </html>{{:Team:Heidelberg/Templates/Del_week14_AE}}<html>
+                                       </html>{{:Team:Heidelberg/Templates/Delftibactin_week20}}<html>
                                  </p>
                              </div>
-                            <div class="tab-pane" id="c14">
-                                <p style="font-size:12pt; text-align:justify;">
-                                </html>{{:Team:Heidelberg/Templates/Del_week14_AF}}<html>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="d14">
-                                <p style="font-size:12pt; text-align:justify;">
-                                </html>{{:Team:Heidelberg/Templates/Del_week14_AG}}<html>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="f14">
-                                <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week14_G}}<html>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="g14">
-                                <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week14_OP}}<html>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="j14">
-                                <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week14_FG}}<html>
-                                </p>
-                            </div>
                          </div>
                      </div>
@@ Line 745: / Line 299: @@
              </div>
-             <!--Week 15-->
+            <!--Week21-->
              <div class="labjournal-weekly">
                  <div>
                      <ul class="nav nav-tabs">
-                       <li class="active"><a href="#a15" data-toggle="tab">Overview</a></li>
+                       <li class="active"><a href="#b21" data-toggle="tab">Lab book</a></li>
-                      <li><a href="#b15" data-toggle="tab">DelA-E</a></li>
-                      <li><a href="#c15" data-toggle="tab">DelA-F</a></li>
-                      <li><a href="#d15" data-toggle="tab">DelA-G</a></li>
-                      <li><a href="#e15" data-toggle="tab">DelE-F</a></li>
-                      <li><a href="#j15" data-toggle="tab">DelF-G</a></li>
-                      <li><a href="#f15" data-toggle="tab">DelG</a></li>
-                      <li><a href="#g15" data-toggle="tab">DelO-P</a></li>
-                      <li><a href="#h15" data-toggle="tab">DelL</a></li>
-                      <li><a href="#i15" data-toggle="tab">pSB4K5</a></li>
                      </ul>
                  </div>
-                 <div class="jumbotron weekly">
+                 <div class="jumbotron">
-                     <div class="scrollContent nav navbar" data-spy="scroll" data-target="#navbarExample" data-offset="0">
+                     <div class="nav navbar" data-spy="scroll" data-target="#navbarExample" data-offset="0">
-                       <div class="tab-content">
-                            <div class="tab-pane active" id="a15">
-                                <p style="font-size:12pt; text-align:justify;">
-                                </p>
-                            </div>
-                             <div class="tab-pane" id="b15">
+                            <div class="tab-pane" id="b21">
                                  <p style="font-size:12pt; text-align:justify;">
+                                       </html>{{:Team:Heidelberg/Templates/Delftibactin_week21}}<html>
-                                </html>{{:Team:Heidelberg/Templates/Del_week15_AE}}<html>
                                  </p>
                              </div>
-                            <div class="tab-pane" id="c15">
-                                <p style="font-size:12pt; text-align:justify;">
-                                     </html>{{:Team:Heidelberg/Templates/Del_week15_AF}}<html>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="d15">
-                                <p style="font-size:12pt; text-align:justify;">
-                                </html>{{:Team:Heidelberg/Templates/Del_week15_AG}}<html>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="e15">
+                        </div>
-                                <p style="font-size:12pt; text-align:justify;">
+                    </div>
-                               </html>{{:Team:Heidelberg/Templates/Del_week15_EF}}<html>
+                </div>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="f15">
-                                <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week15_G}}<html>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="g15">
-                                <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week15_OP}}<html>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="h15">
-                                <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week15_L}}<html>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="i15">
-                                <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week15_pSB4K5}}<html>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="j15">
-                                <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week15_FG}}<html>
-                                </p>
-                            </div>
-                       </div>
-                   </div>
-               </div>
-            </div>
-             <!--Week 16-->
+            <!--Week22-->
              <div class="labjournal-weekly">
                  <div>
                      <ul class="nav nav-tabs">
-                       <li class="active"><a href="#a16" data-toggle="tab">Overview</a></li>
+                       <li class="active"><a href="#a22" data-toggle="tab">Overview</a></li>
-                       <li><a href="#b16" data-toggle="tab">DelA-F</a></li>
+                       <li><a href="#b22" data-toggle="tab">Lab book</a></li>
-                      <li><a href="#d16" data-toggle="tab">DelA-G</a></li>
-                      <li><a href="#j16" data-toggle="tab">DelF-G</a></li>
-                      <li><a href="#f16" data-toggle="tab">DelG</a></li>
-                      <li><a href="#g16" data-toggle="tab">DelO-P</a></li>
-                      <li><a href="#i16" data-toggle="tab">pSB4K5</a></li>
-                      <li><a href="#z16" data-toggle="tab">Gibson assembly and validation</a></li>
                      </ul>
                  </div>
-                 <div class="jumbotron weekly">
+                 <div class="jumbotron">
-                     <div class="scrollContent nav navbar" data-spy="scroll" data-target="#navbarExample" data-offset="0">
+                     <div class="nav navbar" data-spy="scroll" data-target="#navbarExample" data-offset="0">
-                       <div class="tab-content">
-                            <div class="tab-pane active" id="a16">
-                                <p style="font-size:12pt; text-align:justify;">
-                                </p>
-                            </div>
-                             <div class="tab-pane" id="b16">
+                        <div class="tab-content">
+                             <div class="tab-pane active" id="a22">
                                  <p style="font-size:12pt; text-align:justify;">
-                                     </html>{{:Team:Heidelberg/Templates/Del_week16_AF}}<html>
+                                  </html>{{:Team:Heidelberg/Templates/Delftibactin_overview22}}<html>
                                  </p>
                              </div>
-                             <div class="tab-pane" id="d16">
+                             <div class="tab-pane" id="b22">
                                  <p style="font-size:12pt; text-align:justify;">
+                                       </html>{{:Team:Heidelberg/Templates/Delftibactin_week22}}<html>
-                                </html>{{:Team:Heidelberg/Templates/Del_week16_AG}}<html>
                                  </p>
                              </div>
+                        </div>
-                             <div class="tab-pane" id="f16">
+                    </div>
-                                <p style="font-size:12pt; text-align:justify;">
+                </div>
-                               </html>{{:Team:Heidelberg/Templates/Del_week16_G}}<html>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="g16">
-                                <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week16_OP}}<html>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="i16">
-                                <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week16_pSB4K5}}<html>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="j16">
-                                <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week16_FG}}<html>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="z16">
-                                <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Template/Del_week16_Gibson}}<html>
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Team:Heidelberg/Delftibactin/Delftibactin

From 2013.igem.org

Latest revision as of 02:19, 5 October 2013

Team

Project

Notebook

Parts

Software

Human Practice

Safety

Modeling

Delftibactin. Bringing it all together.

Week 1

Week 3

Week 15

Week 19

Week 20

Week 21

Week 22

Week 23

Graphical Abstract

29-04 - 05-05-13

Reproduction of Johnsson et al. Paper

Aim

Preparations

Gold Precipitation on ACM Plates

Result

Gold Precipitation in D. acidovorans Supernatants

Result

Gold Precipitation in D. acidovorans Supernatants at different Gold Chloride Concentrations

Result

13-05 - 19-05-13

Gold Recovery from Electronic Waste

Precipitation of Gold from Electronic Waste

Protocol

Result

05-08 - 11-08-13

Melting Gold Nanoparticles to Solid Gold

02-09 - 08-09-13

Optimizing Growth Conditions of D.acidovorans and Evaluation of Endogenous Background Gold Precipitation

On Agar Plate following ON Incubation at 30°C

Result

09-09 - 15-09-13

Optimizing Growth Conditions of D.acidovorans and Evaluation of Endogenous Background Gold Precipitation

On Agar Plate following 3 Day Incubation at 30°C

Result

On Agar Plate following 3 Day Incubation at RT

Result

In ACM Medium following 2 Day Incubation at 30°C

Result

In ACM Medium following 3 day Incubation at 30°C

Result

Purification of Delftibactin

Purification

Testing Purified Delftibactin

Micro-TOF

Result

Triple Clone pIK8.6, DelH and DelH I 6B

Preparations

Electroporation

Preparation of Cultures for Purification and MICRO-TOF

Purification of Delftibactin

MICRO-TOF

Result

17-09 - 22-09-13

Triple Clone pIK2.6, DelH and DelH I 6B

Electroporation

Preparation of Cultures for Purification and MICRO-TOF

Purification of Delftibactin

MICRO-TOF

Result

23-09 - 29-09-13

Evaluation of Background Expression and Indcibility of Possible Target Strains

Electroporation of mRFP Controls

Result

New BL21 DE3

Electroporation of mRFP Controls

Result

Electrocompetent E. coli BL21 DE + DelRest and pIK8.6

Electroporation of DelRest, DelH and pIK8.6