Team:Bielefeld-CeBiTec/Notebook/PlasmidDisplay
Plasmid Repressor Interaction Assay
- 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
- Purification of his-tagged lacI to achieve higher purity it was performed automatically with an FPLC from BioRad.
- Plasmid isolation of BBa_R0010 and BBa_K592008 as a midiprep to generate enough material for our interaction assays.
- 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.
- MS-Analysis of SDS-Page bands
- 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 tubes were centrifuged with 1000 g for 1 minute.
- The supernatant after centrifugation was stored for analyzing protein and DNA amounts of the samples.
- The DNA amount of the supernatant after centrifugation was analyzed via nanodrop and the protein amount analyzed via SDS-PAGE.
- As negative controls we did not add protein to the agarose column in one sample and no plasmid in another sample.
- We tested the interaction of lacI with the lac promoter and lac operator.
- We also performed the complex formation at 0 °C and 37 °C
- Steps:
- 25 µL Ni-NTA agarose was put in a reaction tube. Then the sample was centrifuged.
- The agarose column was washed three times with potassium phosphate (Kpi) buffer.
- 10 µg protein (we used lac Repressor with a His-Tag as model protein) in 20 µL Kpi buffer was added and incubated for 30 min. Then the sample was centrifuged.
- The column with the protein was washed three times in Kpi Buffer (Volume: 27 µL). Then the sample was centrifuged.
- 1,5 µg plasmid (lacO) was mixed with 20 µL binding buffer and incubated with the column for 15 minutes. Then the sample was centrifuged.
- Unbound DNA was washed away three times with a binding buffer.
- 3x elution with binding buffer and analytes (for this experiment: IPTG).
- Imidazol was used to release proteins from the agarose.
- Unfortunately the DNA concentration could not be measured by the nanodrop. 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
- The Standard protocol for suffix insertion was followed. Unfortunately unsuccessful
- 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 at which the formation of the DNA-Protein Komplex was performed.
- Not adding protein to the agarose column is a good negative control.
- The rest of the agarose column after the final step was dissolved in 10 µL water and also used for the analysis via gelelectrophoresis.
- Verification of protein amount in the supernatant of every step in the assay.
- Detection via SDS-PAGE
- No protein detected, except in the step where it was released with imidazole. this means that the signal we detect is due to release of the plasmid, not due to loss of protein.
- 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.
- Small effect of the different IPTG concentrations 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 for a 3A Assembly
- Coating of an ELISA plate, to test if the protein can still perform its function if it is unspecifically immobilized
- Preparation of an electrophoretic mobility shift assay (EMSA): PCR for the creation of Cy3 labeled DNA fragments containing the lac operator, lac promoter, just T7 and RBS as a negative control and the ars operator.
- Interaction study of lacI-lacO via EMSA
- We performed an electrophoretic mobility shift assay with lacO and lacI
- A shift was visible, but no clear band could be seen.
- Repetition of EMSA for lacO-lacI interaction
- We added different amounts of the lacI, to achieve a clear shift of DNA band.
- The gel melted.
- 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, because no plasmid remained on the column and a strong signal was visible in the first elution step.
- Test of PRIA in a 96 well plate for ELISA
- 96 well plate as new system for PRIA
- Unspecific adsorption of lacI.
- No elution of lacO. DNA was unbound.
- The plate was treated with the same procedure as the agarose column.
- Analysis by SDS Page showed different bands, probably the BSA used for blocking of the ELISA plate was seen as well.
- PRIA with different salt concentrations and complex formation prior to adding the protein to the agarose column.
- We tried to create the complex of lacO and lacI first and then bound it on the agarose column.
- We tested higher salt concentrations (0.5 M, 0.75 M, 1 M, 2 M KCl) in the binding buffer. Moreover, we examined whether we could also utilize sodium chloride.
- Sodium chloride could also used instead of potassium chloride, which could be useful in terms of usability.
- 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 with different protein concentration
- Test to bind DNA in filter paper
- The test was done to examine whether DNA would be washed out of paper.
- 500 ng DNA was applied on a filter paper strip. Then the filter paper was stained with ethidium bromide (EtBr).
- For the negative control, a filter paper strip without DNA was stained with EtBr.
- No detection of DNA possible due to the high intrinsic signal of the paper strip.
- We transformed BioBrick Bba_K1321340 from the distribution. It is a cellulose binding domain (CBD) which is reported to bind strongly to cellulose.
- 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-blcR-sfGFP-His
- pSB1C3-T7-RBS-arsR-CBD
- pSB1C3-T7-RBS-blcR-CBD
- pSB1C3-T7-RBS-lacI-CBD
- Cy3- and amino-labeled lacO operator
- Cy3- and amino-labeled ars operator
- Cy3- and amino-labeled blc promoter
- Coating of a 96-well plate with microcrystalline cellulose.
- The instruction is from the iGEM team 2014 at Imperial university: 40 g cellulose is dissolved in 250 mL water. 200 µL cellulose solution is put in a well and incubated in an incubator (37 °C) overnight. We did a serial dilution to determine the optimal amount of cellulose per well. The optimal amount was 100 µL of a solution with 16 g/L avicell cellulose.
- 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.Fluorescence of the pellets was detected via our special form of photography.
- Preparation of necessary buffers for the activation of Paper.
- Immobilization of DNA on paper
- Whatman filter paper was activated through PDITC dissolved in DMSO. The DMSO was not dried properly, so the PDITC did not dissolve properly.
- Cy3- and amino-labeled DNA was immobilized on the activated paper and could be detected with the Typhoon scanner. It was created by hybridization od an amino labeled oligo and a Cy3 labeled oligo. After washing with water DNA signals were weaker. After washing with SSC buffer the signal vanished completly due to the denaturating effects of SSC.
- 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.
- Quantification of protein
- Concentrations of the cell lysates with the fusion proteins were determined with Rotiquant.
- An SDS PAGE was performed to check if the fusion proteins with a CBD were overexpressed. Unfortunately an overexpression could not been seen clearly.
- Immobilization of DNA
- PDITC was dissolved in ethanol and acetic acid, and used for activation of Paper. No washing with SSC buffer
- The oligos that are to be immobilized were in PCR buffer or 50 mM sodium phosphate buffer, pH 8.0. The oligos in the 50 mM sodium phosphate buffer performed better and were more resistant to washing.
- We blocked the membranes with the immobilized DNA with milk powder or BSA and
- Immobilization of protein
- We planned to add the CBD fusion proteins to paper and wash the unbound protein off
- Test for the visualization of protein on paper: Coomassie staining solutions and destaining solutions normally used for SDS PAGES worked just fine and stained the protein spots on paper specifically.
- The proteins bound strongly and unspecific to paper. We tested various buffers to remove them and washed over night. Nevertheless no diminishing of the coomassie signal was detected.
- EMSA to check arsR for binding to DNA, successful clear band shift.
- cloning of an T7-RBS-UTR-sfGFP-His device
- Purification of his tagged arsR and analysis via SDS-Page
- Immobilized DNA in 96 well plate
- DNA was immobilized on the 96 well plate following the same protocol as for normal paper
- 100 µL of the protein solution with 20 µg/ml were added to each well. The proteins were the GFP tagged fusion proteins.
- the wells were washed with 200 µL of KBT buffer
- 90 µL of sample solution with a final concentration of 100 µg/ml was added.
- Immobilzed DNA on Paper
- We were able to detect the immobilized Cy3 labeled DNA and the GFP tagged Fusion proteins with an Ettan DIGE fluorescence scanner.
- PRIA and EMSA for GFP tagged proteins was performed
- The formation of the protein DNA complex was proven by both methods, nevertheless no release of DNA could be measured.
- PRIA was repeated with conditions similar to those of EMSA
- EMSA was performed to check whether a reaction to the analytes GBL, GHB or SSA could be seen for blcR
- We tested if our sfGFP arsR fusion protein would show FRET when interacting with the Cy3 labeled arsO. This was not the case.
- The detection limit for GFPtagged proteins and Cy3 labeled DNA was determined by scanning of an serial dilution on paper in the Ettan DIGE
- By washing we determined the durability of the immobilized DNA on paper. Their was no difference between paper activated with PDITC dissolved in EtOH and PDITC dissolved in DMSO dried with molecular sieves. Therefore we recommend the activation of paper with PDITC dissolved in EtOH, which is easily available. Furthermore, the activated paper can be used up to two weeks after activation for the immobilization of DNA.
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