Team:Tuebingen/Experiments

PacMn

Experiments

Phase 1: Cloning

After designing our constructs and the primers for Gibson Assembly (GA), we ordered the DNA molecules from Integrated DNA Technologies, Inc. (IDT). The first step in cloning was to prepare the individual fragments for GA. This consisted in amplifying each fragment with the special primers designed to add complementary overhangs, and linearizing the vector backbone through restriction digestion. The products were qualitatively analyzed by gel electrophoresis and their concentrations (ng/µl) were measured with a spectrophotometer (Fig. 1).
Figure 1: Overview of fragment preparation for Gibson Assembly.

Figure 1: Overview of fragment preparation for Gibson Assembly.

With the ready-to-use fragments, the next step was to assemble them. The assembly itself only takes about an hour, but the whole process from assembling to having a confirmed positive clone required six main steps (Fig. 2):
  1. Gibson Assembly: Assembling the fragments in vitro.
  2. Transformation: Transforming chemically competent E. coli with DNA from the assembly reaction.
  3. Colony PCR: Preliminary screening of potential positive transformants by amplifying insert DNA and analyzing its size.
  4. Plasmid isolation: Extraction of plasmid DNA of potential positive transformants for further analysis.
  5. Restriction digestion: Linearization of isolated plasmid DNA for a second size analysis (of the entire plasmid).
  6. Sequencing: Sending the size-confirmed candidates for sequencing to rule out mutations within the insert DNA.
Figure 2: Schematic overview of cloning workflow. The main steps are ordered from 1 to 6.

Figure 2: Schematic overview of cloning workflow. The main steps are ordered from 1 to 6.

Phase 2: Testing

The most interesting test for our constructs consisted in measuring fluorescence in the presence of different manganese(II) chloride concentrations (Fig. 3). The measurements for all constructs were conducted simultaneously in a plate reader, including an empty pUC19 vector as a control.
Figure 3: Schematic overview of fluorescence measurement workflow. The main steps are ordered from 1 to 3.

Figure 3: Schematic overview of fluorescence measurement workflow. The main steps are ordered from 1 to 3.

As a qualitative test for fluorescence, we analyzed our different clones via fluorescence microscopy. We also observed the morphology of our cells containing the different constructs to search for stress phenotypes (Fig 4.).
Figure 4: Analysis of cell fluorescence and morphology via Microscopy.

Figure 4: Analysis of cell fluorescence and morphology via Microscopy.

As a qualitative validation of our Mn-inducible components, we grew E. coli containing the chromoprotein gene on selective solid and liquid LB medium with manganese(II) chloride (Fig. 5).
Figure 5: Testing the effect of chromoprotein expression on cell color.

Figure 5: Testing the effect of chromoprotein expression on cell color.

To roughly test the transgenerational propagation stability of our constructs, we performed a sub-cultivation experiment on non-selective solid LB medium (Fig. 6).
Figure 6: Sub-cultivation experiment as a plasmid stability test.

Figure 6: Sub-cultivation experiment as a plasmid stability test.

To analyze the functionality of our manganese-inducible promoters in the absence of protein expression, we performed Reverse Transcriptase (RT)-PCR. Here, we evaluated the transcriptional activation presumably initiated by manganese(II) chloride (Fig. 7).
Figure 7: Verification of mRNA synthesis via RT-PCR.

Figure 7: Verification of mRNA synthesis via RT-PCR.

Image sources

Figures 1-7: Created with BioRender.com