Using Tetrameric Lac Repressor to Improve the Autogenously

The lac repressor construct we used has the 11 C-terminal residues of the lac repressor truncated to create a dimeric repressor. While we have used a dimeric repressor for clarity of understanding and studying allostery in Chapters 3 and 4, here we just want a more effective switch. With that goal in mind we sought to restore the tetramerization domain and multiple operator DNA sequences to restore the allosteric of lac repressor to two DNA operators.

First, we cloned a second Lsym operator DNA sequence 92 base pairs downstream of the first Lsym site (Figure 5.13). We did not necessarily expect this downstream site to be able to directly regulate transcription. We transiently transfected dimeric lac repressor with one Lsym operator (pSW-Luc) and dimeric lac repressor with two Lsym opeartors (pSW-Luc Lsym x2) into ARPE-19 cells and assayed for luciferase with and without IPTG (Figure 5.14). The addition of a second operator decreased both leakiness and maximal expression.

Figure 5.13. The pSW-Luc Lsym x2 plasmid. This plasmid expresses an autogenously regulated dimeric lac repressor construct which can bind two Lsym operator sites and has luciferase as a readout.

Figure 5.14. Effect of adding of a second Lsym operator with dimeric lac

repressor. The pSW-Luc construct expresses dimeric lac repressor and has a single Lsym operator DNA. The pSW-Luc Lsym x2 construct has a second Lsym operator DNA site 92 base pairs downstream. The addition of this second operator decreases

Next, the 11 C-terminal residues were re-inserted into the EuLac gene to make what we will call EuLacTet to make the pSW2-Luc/GFP plasmids (Figure 5.15). This gene still has the 11 amino acid linker and NLS sequence following the full lac repressor itself, not to mention the partial 2A protein appended to the C-terminus. We first wanted to see if the EuLacTet protein could form tetramers after 2A breakage. We cloned the

EuLacTet with a 6x Histidine tag (His-tag) into our standard pBAD expression vector and followed the standard lac repressor purification protocol. We also purified the original construct without the 11 C-terminal insertion which should be dimeric. These putative dimeric and tetrameric proteins were run on a native electrophoresis gel where

oligomeric state will be preserved and stained for protein. The native gel confirmed that the tetrameric protein ran at twice the molecular weight of the dimeric protein (Figure 5.16).

Figure 5.15. The pSW2-Luc plasmid. This plasmid expresses an autogenously

regulated tetrameric lac repressor construct which can bind two Lsym operator sites and has luciferase as a readout. Two DNA operators are known to allosterically bind

tetrameric lac repressor to increase the operator binding affinity and decrease repression. One model of this behavior is through DNA loops (pictured) wherein the spacing between the two operator DNA sites is critical.

Figure 5.16. Native gel electrophoresis to discern oligomerization state. The EuLac construct which lacks 11 C-terminal residues known to form a coiled-coil domain has two primary bands near ~84 kDa and ~42 kDa which correspond to dimeric and monomeric species, respectively. The EuLacTet construct, which has the 11 C-terminal residues restored, forms a single tight band near the predicted ~174 kDa which

corresponds with a tetrameric species. The highest band in both wells is a

contamination from the purification. Both constructs are oligomerizing as designed. We transiently transfected tetrameric lac repressor with two Lsym operators (pSW2-Luc) into ARPE-19 cells and assayed for luciferase with and without IPTG (Figure 5.17). The addition of the tetramerization domain decreased leakiness and increased maximal expression over the dimeric construct. Overall however, the potential cooperative effect of tetrameric lac repressor binding to two operators was less than has typically been seen in E. coli. This construct has an approximately 3-3.5-fold induction with IPTG and the leakiness is decreased to ~25% of constitutive expression. These are both significant gains from the starting pSW-Luc construct, however it does not appear

that it is necessarily due to the allosteric advantages of binding two operators with tetrameric lac repressor.

Figure 5.17. Effect of adding of a second Lsym operator with tetrameric lac repressor. The pSW2-Luc construct expresses tetrameric lac repressor and has two Lsym operator DNA sequences. The decreased leakiness is maintained (lower right green arrow) and the maximal expression is marginally improved (upper right green arrow). Overall a significant improvement from the starting pSW-Luc construct has been obtained.

To measure if the tetramerization is playing a significant role we sought to repeat a classic experiment from E. coli. It is known that the spacing between the two operator DNA sites critically effects the ability of lac repressor to regulate gene expression resulting in a periodicity of optimal spacing (Müller et al., 1996). Optimal spacing was found at 70.5, 81.5 and 92.5 base pairs with significant drop offs in repression by altering the spacing even 1 or 2 base pairs. With this in mind we inserted or removed one or two base pairs between the two operator sites and also changed to spacing entirely to 70 base pairs. We tested these constructs in transient transfections of ARPE-19 cells with

and without IPTG (Figure 5.18). There was no significant difference in measured

induction ratios between these constructs. Either the cooperative effect is minimal in cell culture or further construct optimization is needed.

Figure 5.18. Alternate Lsym operator DNA base pair spacings. It has been observed in E. coli that optimal repression by tetrameric lac repressor occurs when the two

operator sequences are properly spaced. We found no significant change in induction ratio with alternative spacings.

We did reduce leakiness and increase dynamic range by using tetrameric lac repressor and two operator DNA sites. It is unclear however if the system is working as has been previously reported in bacteria.

5.6 Using Lac Repressor Mutations to Improve the Autogenously Regulated