• 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
#Probeauftragung+Bild?
• 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

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