Biological Experiments
In this section, we present a general overview of our performed biological experiments during the past months. For a detailed insight in our daily lab work feel free to take a look at the PDF version of our lab notebook below or at our Experiments.
- PCRs for genes of ATF2 (BBa_K5317016), CcpA (BBa_K5317014) GraR (BBa_K5317015) and MTF1.
- Prepared PCR reactions for metal-gene cloning (MTF-1, mRuby2 (BBa_K5317001)).
- Restriction digestion of pEGFP-C2 (BBa_K3338020) plasmids for MRE promoter constructs using AseI and NheI enzymes.
- HiFi reactions of MRE promoter constructs (MREdada, MREa, MREnormal, MREd).
- Colony PCR and MiniPreps for analyzing MRE constructs.
- Sequencing of MRE promoter constructs.
- Repeated PCRs from gBlocks for the genes of PknB, CcpA, and GraR.
- PCRs for genes of ATF2, CcpA, and GraR (mRuby2 constructs).
- Restriction digestion with DpnI of GraR and CcpA.
- Restriction digestion of pEGFP-C2 with BamHI and NheI for creating a backbone for HiFi reaction of CcpA, GraR and MTF1.
- HiFi reactions for various constructs:
- PknB and pEGFP-C2 to composite part CMV-EGFP-PknB (BBa_K5317018).
- CcpA, mRuby2 and pEGFP-C2 to composite part CMV-CcpA-mRuby2 (BBa_K5317019).
- GraR, mRuby2 and pEGFP-C2 to composite part CMV-GraR-mRuby2 (BBa_K5317020).
- MTF1, mRuby2 and pEGFP-C2 to composite part CMV-MTF1-mRuby2 (BBa_K5317012).
- Heat shock transformation of cloned HiFi products.
- Colony PCR for CcpA, GraR, and MTF-1 (mRuby2 constructs).
- MiniPreps and sequencing of CcpA, GraR, and MTF1 (mRuby2 constructs).
- Restriction digestion of pEGFP-C2 with BamHI & SalI for PknB construct (repeated multiple times).
- Restriction digestion of pEGFP-C2 with EcoRI & NheI for MTF1 construct (repeated multiple times).
- Repetition of HiFi reaction for CMV-EGFP-PknB and CMV-MTF1-mRuby2.
- Heat shock transformation of HiFi products.
- Colony PCRs, MiniPreps and sequencing for the composite parts.
- HiFi reaction of ATF2, mRuby2 and pEGFP-C2 to composite part CMV-ATF2-mRuby2 (BBa_K5317021).
- Heat shock transformation of HiFi product.
- Colony PCRs, MiniPreps and sequencing of the composite parts.
- Repetition of PCR for PknB with Phusion- and Q5-Polymerase, followed by another HiFi reaction for CMV-EGFP-PknB.
- Heat shock transformation and colony PCR, MiniPrep and sequencing for CMV-EGFP-PknB.
- Repetition of HiFi reaction and following Colony PCR of ten clones for CMV-EGFP-PknB.
- Success for one clone (K6) validated via gel electrophoresis.
- Mini-Prep & Sequencing of clone K6.
- Further sequencing of K6 to validate correctness.
- Showed mutation in the sequence of PknB.
- Order of new gBlock of PknB.
- Thaw 2x 1 ml HEK293T P5 cells.
- Passaging of HEK293T cells.
- First test transfection of HEK293T cells with CMV-MTF1-mRuby2 and pEGFP-C2.
- Repetition of HiFi, colony PCR, MiniPrep and Sequencing of CMV-EGFP-PknB with new PknB gBlock (successful).
- Qualitative validation of single MTF-1, MREwt, MREa, MREd and MREdada as well as the co-transfection of MTF-1 with all promoter constructs.
- Qualitative validation of single PknB, GraR, ATF2 and CcpA.
- Kill curve of HEK293T cells under treatment with CuCl2 (0 µM, 50 µM, 250 µM, 500 µM, 1 mM, 2 mM) and ZnCl2 (0 µM, 50 µM, 250 µM, 500 µM, 1 mM, 5 mM).
- Qualitative validation of single ATF2 and CcpA.
- Qualitative validation of PknB co-transfected with ATF2 and GraR.
- Kill curve of HEK293T cells under treatment with CuSO4 (0 µM, 50 µM, 250 µM, 500 µM, 1 mM, 2 mM).
- Qualitative validation of MTF-1 co-transfected with MREwt and MREdada promoters via fluorescent microscopy before and after stimulation with CuSO4.
- Qualitative validation of MTF-1 co-transfected with MREa and MREd promoters via fluorescent microscopy before and after stimulation with CuSO4.
- Restriction digestion of pEGFP-C2 with AseI & BamHI for creating a backbone for the HiFi reaction of ATF2.
- Quantitative FACS validation of metal constructs after stimulation with CuSO4.
- Cloning of ATF2_miRFP-Promoter using the Parts miRFP670 (BBa_K5317002) and ATF2-3xCre3xAP1-Promoter (BBa_K5317017) via HiFi DNA Assembly and heat shock transformation in E. coli DH5-alpha.
- Repetition of ATF2_miRFP-Promoter (BBa_K5317022) cloning after failure in the previous week.
- Sequencing of correct ATF2_miRFP-Promoter clone K7.
- Qualitative validation of antibiotic constructs via fluorescent microscopy.
- Kill curve of HEK293T cells under treatment with Ampicillin (0 µg/mL, 5 µg/mL, 10 µg/mL, 25 µg/mL, 50 µg/mL, 100 µg/mL, 500 µg/mL)
- Testing of different construct combinations (PknB + Promoter; ATF2 + Promoter; single Promoter; PknB + ATF2 + Promoter).
- Qualitative and Quantitative validation of antibiotic constructs via fluorescent microscopy and FACS analysis, respectively.
- Further qualitative validations of metal and antibiotic constructs via fluorescent microscopy.
