Part:BBa_K4238007

ADH1p+loxP+mCherry+CYC1t+loxP+mNeonGreen+ADH1t

Description

This part was created to assay the recombination by the Cre-loxP system in yeast. Cre is an enzyme that performs site-specific recombination and can be used to delete a part of sequences irreversibly. [1][2] Cre has the enzymatic activity of circularly excising the region flanked by recognition sequences called loxP. Two Cre proteins recognize and bind to the loxP site, forming a dimer. The two Cre-loxP dimers attach so that the two loxP sequences face in opposite directions to form a tetramer. Finally, the DNA is cleaved at the center of the loxP site, causing recombination by crossover (Figure 1). This can be used to remove DNA sequences between two recognition sequences.

FIgure 1 The mechanism of recombination.

When Cre is not present in the cell, mCherry (red fluorescent protein) flanked by loxP is expressed, while mNeonGreen (green fluorescent protein) in the back is not expressed because of the CYC1 terminator. On the other hand, if Cre is present in the cell, only mNeonGreen is expressed because the mCherry sequence is cut out. Therefore, the recombination timescale and other parameters can be evaluated by observing the fluorescence color under a microscope.

Wet results

The fluorescence of mCherry and mNeonGreen in yeast transformed with only BBa_K4238007 (control, Figure 2) and yeast transformed with BBa_K4238006 in addition to BBa_K4238007 (test, Figure 3) were compared by microscopic observation. The samples used for microscopic observation were inoculated on YPD medium in the case of control and on SC-U medium in the case of test, and were observed with setting of transmitted light (exposure time 50 ms), TxR (wavelength 560 nm, exposure time 500 ms), and GFP (wavelength 470 nm, exposure time 1000 ms). The results are shown in Figures 4-6 below.

Figure 2. The completed sequences for the control.

Figure 3. The completed sequences of the test.

Figure 4. Scatter plots of mCherry and mNeonGreen luminance values in control and test cells; the two stars represent the mean value of each group (left: test, right: control). 428 cells of control and 260 cells of test were observed.

Figure 5. Distribution of mCherry fluorescence brightness for control and test.

Figure 6. Distribution of mNeonGreen fluorescence brightness for control and test.

The scatter plots show that the fluorescence brightness of both mCherry and mNeonGreen decreased when cells were transformed with Cre. However, when looking at the distribution of each fluorescence protein, mCherry was originally expressed by most cells and stopped being expressed when Cre was transformed, while mNeonGreen was originally not expressed by most cells and that did not change even after Cre was transformed. This suggests that recombination was successfully performed and mCherry was cut out, but mNeonGreen was not expressed for some reason. This might be because an extra sequence between the promoter and mNeonGreen affected the expression level, or mNeonGreen did not work in S. cerevisiae because it derives from S. pombe. If this inference is correct, then we have confirmed that the recombination of Cre and loxP proceeds satisfactorily under the conditions of this experiment.

References

[1] Siegel, R. W., Jain, R., & Bradbury, A. (2001). Using an in vivo phagemid system to identify non-compatible loxP sequences. FEBS letters, 505(3), 467-473. doi: 10.1016/s0014-5793(01)02806-x
[2] Suzuki, T., & Kubota, H. (2017). First Mathematical Models and Simulations for Use in the Life Sciences. Experimental Medicine Extra, 35(5), Yodosha.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]