• Heat shock transformation of Biobricks needed for further assays was performed. Glycerol stocks were generated and the plasmids isolated according to the kits manual. The relevant Biobricks were:


• BBa_K525998: T7 promotor and RBS
• BBa_C0012: lacI repressor with LVA Tag
• BBa_R0010: lac Promotor
• BBa_K592008: lac Operator


• Furthermore a plasmid with a gene coding for a histagged lacI Protein was transformed, which was kindly provided by Stefan Hoffmann.

• Purification of his-tagged lacI
• Test digest of transformed Biobricks: all clones as expected!
• Plasmid isolation of BBa_R0010 and BBa_K592008

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• Verification of lacI-His
• SDS-PAGE for analyzing the purifity of purified lacI-His
• lacI-His was expected in the 38 kDa bands
• Bands (size 38 kDa) were cut out and destained.

• Identification of the bands cut before
• Tryptic digestion of the proteins from the bands.
• Measurement of the digested proteins via MALDI-TOF/TOF.
• The measured peptide spectre was compared with spectre of other proteins with an E. coli database.
• LacI identified (Mascot score: 765).

• Development of the plasmid repressor interaction assay (PRIA)
• After every step the reaction vessel were centrifuged with 1000 g for 1 minute. The DNA amount of the supernatant after centrifugation was analyzed via nanodrop.
• As negative controls we did not add protein to the agarose in one sample and no plasmid in another sample.
• We tested the interaction of lacI wit lac promoter and lac operator.
• We also performed the assay at 0 °C and 37 °C
• Steps:
• 25 µL Ni-NTA agarose was put in a reaction vessel. Then centrifuge.
• The agarose was washed three times with Kpi buffer.
• 10 µg protein (we used lac Repressor with a His-Tag as model protein) in 20 µL Kpi buffer were added and incubated for 30 min. Then the agarose were centrifuged.
• The agarose was incubated three times in Kpi Buffer (75 µL).
• 1,5 µg plasmid (lacO) was mixed with 20 µL binding buffer and incubated for 15 minutes.
• Unbound DNA was washed away three times.
• 3x elution with binding buffer and analytes (for this experiment: IPTG)
• Imidazol was used to release proteins from the agarose.
• No results! DNA concentration could not be measured. Next time: analysis with gelelectrophoresis.

• Plasmid isolation of E. coli KRX with pSB1C3-lacO
• Concentration: 186,3 ng/µL
• Suffix insertion: lacI-LVA as insert and pSB1C3-T7-RBS as backbone
• Optimizing PRIA
• The DNA amount of the supernatant after centrifugation was analyzed via gelelectrophoresis.
• Repetition of the assay performed before
• In the figure we could detect DNA after staining in ethidium bromide.
• A high DNA amount was unbound.
• Elution with IPTG possible. DNA could be detected after adding the analyte solution to the agarose.<
• We did not see any difference between the temperatures.
• Not adding protein to the agarose is a good negative control.
• The rest of the agarose after the final step was dissolved in 10 µL water and also used for the analysis via gelelectrophorese.
• Verification of protein amount in the supernatant of every step in the assay.
• Detection via SDS-PAGE
• No protein detected.

• Testing analyte solution with different concentrations
• We wanted to test 0.05 mM, 0.5 mM and 5 mM IPTG mixed with binding buffer as elution buffer.
• No effect of the different IPTG concentration to the eluted DNA amount.
• We standardized our assay. Every step took 15 minutes and the volume of the added solutions are 50 µL.
• Restriction of pSB1A3, pSB1C3-lacI-LVA and pSB1C3-T7-RBS

• Interaction study of lacI-lacO
• We did an electrophoretic mass shift assay with lacO and lacI
• It did not work. Next time: titration of the plasmid.

• Repetition of EMSA for lacO-lacI interaction
• Optimizing PRIA with different salt concentrations
• Concentrations 100 mM, 250 mM and 500 mM KCl in binding buffer were tested in PRIA.
• Using binding buffer with 500 mM KCl was best for PRIA.
• 96 well plate as new system for PRIA
• Unspecific binding of lacI.
• No elution of lacO. DNA was unbound.

• PRIA with different salt concentrations and creating a complex formation first before adding to the agarose
• We tried to create the complex of lacO and lacI first and then bound it on the agarose.
• We tested higher salt concentrations (0.5 M, 0.75 M, 1 M, 2 M KCl) in binding buffer. Moreover, we examined whether we could also utilize sodium chloride.
• Cloning of pSB1C3_T7-RBS-lacI-His
• Transformation of pSB1C3_T7-RBS-lacI-Histag in E. coli KRX was successful. Colonies were used to inoculate overnight cultures.

• Repetition of EMSA
• Test to bind DNA in filter paper
• The test was done to look whether DNA can be washed out.
• 3 µL DNA was applied on a filter paper strip. Then the filter paper was stained with GelRed.
• For the negative control, a filter paper strip without DNA was stained with GelRed.
• No detection of DNA possible. Reason for no detection was that the intrinsic signal of the strip was too high.
• We transformed BioBrick Bba_K1321340 from the distribution.
  • Transformation of Biobrick Bba_K1321340 in E. coli KRX
  • Colony PCR of 5 clones on the agarplate after transformation.
  • 5 colonies were used to inoculate overnight cultures.
  • Plasmid isolation
  • Digestion with EcoRI and PstI for screening
• We wanted to develop two new assays.
• One assay is based on GFP labeled protein and immobilized DNA (with Cy3 and amino label) on filter paper. The other one is based on immobilized protein (contains a cellulose binding domain) and Cy3 labeled DNA.
• Primer design for cloning CBD and sfGFP to the devices arsR, blcR and lacI.
• These are the devices we wanted to clone:
• pSB1C3-T7-RBS-lacI-sfGFP-His
• pSB1C3-T7-RBS-arsR-sfGFP-His
• pSB1C3-T7-RBS-lacI-sfGFP-His
• pSB1C3-T7-RBS-arsR-CBD
• pSB1C3-T7-RBS-blcR-CBD
• pSB1C3-T7-RBS-blcR-CBD
• Cy3- and amino-labeled lacO
• Cy3- and amino-labeled arsR
• Cy3- and amino-labeled blc promoter

• We put cellulose on a 96-well plate.
• The instruction is from the iGEM team 2014 at Imperial university: 40 g cellulose is dissolved in 250 µL water. 200 µL cellulose solution is put in a well and incubated in an incubator (37 °C) overnight. We did a serial solution for having different concentration of cellulose on the plate.
• Cloning the devices for our new assays.
• We ran several PCRs for cloning the devices mentioned before and extracted DNA out of gel.
• We assembled the DNA through Gibson Assembly and transformed the assembled DNA in E. coli KRX.
• Clones are evaluated through colony PCR. We cultivated several clones which contained the plasmid.

• Plasmid isolation of the overnight cultures
• All devices were sent to sequencing.
• Cultivation
• E. coli KRX with the plasmid pSB1C3-T7-RBS-arsR-sfGFP, pSB1C3-T7-RBS-lacO-sfGFP-His and pSB1C3-T7-RBS-blcR-sfGFP were cultivated and harvested to check whether the fusion proteins could be functionally expressed.

• Immobilization of DNA on paper
• Whatman filter paper was activated through PDITC dissolved in DMSO.
• Cy3- and amino-labeled DNA was immobilized on the activated paper and could be detected with the Typhoon scanner. After washing DNA signals were weaker. The reason could be that PDITC could not be dissolved in paper, that's why we wanted to dissolved the DNA in ethanol and acetic acid next time.
• Cultivation
• E. coli KRX with the plasmid pSB1C3-T7-RBS-arsR-sfGFP, pSB1C3-T7-RBS-lacO-sfGFP-His and pSB1C3-T7-RBS-blcR-sfGFP were cultivated and were disrupted via sonification.

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