Team:Heidelberg/Delftibactin/Delftibactin

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                        <h1><span style="font-size:180%;color:#FFCC00;">Delftibactin.</span><span class="text-muted" style="font-size:120%"> Bringing it all together.</span></h1>
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                                      <h1>Week 1</h1>

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We obtained D. acidovorans DSM-39 from the ZSMZ 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. 
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                                  <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.  
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                                  <h1>Week 15</h1>
                                  <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">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.  </p>
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                                  <h1>Week 19</h1>
                                  <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. D. 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. </p>
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                                  <h1>Week 20</h1>
                                  <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">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. </p>
                                  
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                                  <h1>Week 21</h1>
                                  <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%;">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>
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                                  <h1>Week 23</h1>
                                  <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. </p>
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