Cloning, expression and characterization of ELPs

To make our bacteria more controllable in a therapy we wanted to prevent them from dividing. We designed a sequence of Elastin-Like Polypeptides (ELPs) that could theoretically form a hydrogel within the cells, which would inhibit division.

We needed to come up with a way to crosslink the system to make the interactions strong enough to form a stable hydrogel. There were two strategies that we wanted to try and they involved constructing a triblock ELP containing a (relatively) hydrophilic core sequence, sandwiched by two hydrophobic outer sequences. These sequences can be seen below. To the end of each hydrophobic block, a crosslinking group is attached differing for both strategies. We wanted to include and test both methods to have a higher chance of succeeding. We used Leucine zippers Z1 and Z2 and rapamycin binding domains. A schematic representation of what the constructs look like can be seen in Figure 1. All the different parts of the constructs can be seen in Table 1.

First, existing ELP gene sequences that were already present at our university and provided to us by Dr. Le, were digested and ligated to create two constructs with different hydrophilic linkers of which the gels can be seen in Figure 2, as described in more detail in Week 20 of the Notebook. The hydrophobicity of the ELPs is determined by their guest residue as is also described in the Project Description. These linkers contain the guest residues Alanine (A) and Glycine (G), one with 20 repeats of VPGAG_[3]VPGGG_[2] and one with 24 repeats of this same segment. These sequences are called A_[100] and A_[120], respectively. Both of these hydrophilic linkers contain hydrophobic segments at their ends, where the guest residue is Isoleucine (I). The hydrophobic sequence looks as follows: VPGIG_[60], abbreviated as I_[60]. Successful ligation was confirmed by a double digestion with restriction enzymes at either ends of the constructs (Figure 1B). In addition, the plasmids were sequenced by Sanger sequencing (Azenta). Next, our gBLocks (FRB, FKBP12, Z1 and Z2) were cloned into an empty pET24a(+) vector. This was confirmed by colony PCR and sequencing of the plasmids. They were then combined with A_[100] and A_[120] to give us our final constructs that can be seen in Figure 1 and Table 1. Successful ligation was confirmed for each construct through means of sequencing.

After successfully creating the plasmids containing our desired constructs, we expressed them in E. coli BL21(DE3). However, the A_[100] constructs lagged behind with cloning, so only the A_[120] constructs were expressed. Because we managed to form a gel with these constructs, as seen in the Hydrogel Formation paragraph, the decision was made not to express the A_[100] constructs to save time. We then purified all constructs using inverse transition cycling, a process in which the T_t of the ELPs is utilized to separate them from cellular debris and other proteins. Afterwards, dialysis was performed to get rid of any residual salt from the inverse transition cycling. We managed to purify all but one construct, namely FKBP12-A120-FKBP12 (Figure 3). The latter protein was not expressed at all, as evidenced by the gel containing the lysate in Week 28 of the Notebook. Since this was the only protein containing FKBP12 at its N-terminus, this sequence might somehow interfere with the expression. The products that were purified were then freeze-dried and stored at -20 °C until further use. The average yield of the Z1/Z2 constructs was 150 mg/L culture, whereas for the FRB/FKBP12 constructs it was 50 mg/L.

Construct	Expected Molecular weight (kDa)	Number in gel
Z1-A120-Z2	105.1	1
Z2-A120-Z2	105.0	2
FRB-A120-FRB	120.5	3
FKBP12-A120-FKBP12	122.5	4
FRB-A120-FKBP12	121.5	5

Characterization of our in situ gelation within cells with the use of fluorescence recovery after photobleaching (FRAP) microscopy required us to design a model protein which could interact with the hydrogel, and which we could visualize under the confocal microscope. We decided to use VPGIG_[60]-mNeonGreen. . The fluorescenct protein would have a lower diffusion rate after bleaching compared to non-hydrogelation situation. We cloned the gene fragment into a pBAD vector under the control of an arabinose promoter, to allow for orthogonality between this vector and the pET24a(+) vector encoding the zipper-ELP construct. Successful cloning was verified by a double digestion of the plasmids as well as sequencing.

However, to our surprise, the sequencing results indicated that instead of a VGPIG_[60] fragment, the mNeonGreen was attached to a VPGAG_[3]VPGGG_[2][12] fragment, which is hydrophilic, relative to the VPGIG_[60] fragment. This is likely due to a labeling mistake by a previous user of the plasmid. Due to a lack of time, we decided to continue using this construct in our microscopy experiments as a model protein to monitor the diffusion of proteins within the gelated bacteria. The results of our digestion can be seen in Figure 4.

Z1-A120-Z2 and [A3G2]_[12]-mNeonGreen were co-expressed in E. coli BL21(DE3), as is described in week 36 and 37 of the Notbeook. . SDS-PAGE was then used to confirm the presence of both proteins. The results of this can be seen in Figure 5. After some trial and error using different conditions for the co-expression, we managed to express both the ELPs, as well as the fluorescent protein. E. coli containing both proteins were then used for FRAP microscopy of which the results are found here.

The bacteria containing Z1-A120-Z2 and [A3G2]_[12]-mNeonGreen were then used to determine if diffusion throughout the cell was affected by the formation of the gel. This was done by using Fluorescence recovery after photobleaching (FRAP). We hypothesized that once the gel is formed intracellularly, the diffusion rate is negatively affected due to an increase of viscosity of the cytosol. More about this can be read here.