Single Substrate ssrA Degradation Quantification

In order to understand the role of degradation in synthetic gene circuits, it was nec-essary to first quantify the degradation rate of each ssrA degradation tag variant on a single substrate. In our single substrate system, protein degradation due to dilution by cell division and enzymatic degradation of ClpXP combines to contributed to the overall protein degradation rate. Generally, as a cell divides, the overall number of proteins in a cell is divided between the two daughter cells. This degradation due to dilution rate was accurately quantified in our experimental system by measuring the protein disappearance rate of GFP with no ssrA degradation tag attached to the C-terminus of the protein. When the ssrA degradation tag was present, enzymatic degradation also occurred. The ssrA degradation tag directed the GFP to be enzy-matically degraded, specifically by the ClpXP degradation machinery. Changing the last three amino acids of the 11-amino acid tag caused instability in the binding to the ClpX subunit of the ClpXP degradation machinery, thereby slowing recognition of the tag and ultimately decreasing the enzymatic degradation rate.

A set of plasmids was constructed using a pET28 plasmid backbone to analyze the degradation rates of each ssrA tag variant. Each plasmid was constructed with the

Figure 4.2 : General plasmid design for single substrate degradation exper-iments. Single substrate degradation plasmids have a pET28 Kan^R backbone with the IPTG inducible promoter p_L−lac01 regulating the expression of gfp-ssrA.

IPTG inducible promoter p_L−lacO1 promoter, yemGFP (monomeric yeast-enhanced green fluorescent protein), with the ssrA LAA degradation tag variant sequence added to the C-terminus (Fig. 4.2). Once this plasmid was constructed, PCR mutagenesis was used to alter the last three amino acids, to yield the LVA, AAV, and ASV vari-ants. In order to create a plasmid with no degradation tag on GFP (to measure the degradation due to dilution rate by cell growth and division),the T-S linker between the protein and the tag was altered to a SpeI restriction enzyme site using PCR mu-tagenesis. With SpeI sites flanking the degradation sequence and, te ssrA tag wereas tn be reved using SpeI digestion. Then the plasmid was self-ligated to reform a func-tional plasmid.

To create a measurable single substrate system, the IPTG-inducible promoter pLac01 was used to drive the expression of GFP. The ssrA degradation tag was added to the C-terminus of GFP by insertion mutagenesis. Site-directed mutagenesis was used to change the last three amino acids for each variant. After this system was built on a

pET28 high-copy number expression plasmid, it was transformed into JS006 compe-tent cells [5] with LacI integrated back into the genome. Cells transformed with this plasmid system were induced with 2 mM IPTG to activate GFP expression prior to loading on the microfluidic device to ensure a saturating steady state level of fluores-cence. Once loaded, these cells grew in the presence of inducer for an additional two hours, after which the inducer was removed and the decay of green fluorescence was recorded over time. Red fluorescent dye was added to the inducing media to ensure the complete removal of the inducer (Fig. 4.3).

Time-lapse single cell microscopy was used to accurately measure the single sub-strate fluorescence degradation rate of each of the ssrA degradation tag variants.

A custom microfluidic device that wllows rapid switching between inducing media and non-inducing media was used. The custom microfluidic device used a dial-a-wave junction to rapidly and accuratly switch between two distinct medias (Fig. 4.4(A-Red Circle, B)). This transition from inducing media to non-inducing media was nearly in-stantaneous and took less than three minutes for the red dye to no longer be detected (Fig. 4.4(C)). The single substrate plasmid (pET28-GFP-ssrA) was transformed into JS006 LT cells and plated on LB agar + 50 µg/mL of kanamycin. A single colony was selected to inoculate an overnight culture that did not exceed 18 hours. To prepare cells for the microfluidic device, 25 µL of the overnight culture was diluted into 25 mL of LB + 2 mM of IPTG + 50 µg/mL of kanamycin and allowed to grow until an OD600 of 0.15 was reached.

A

B

Figure 4.3 : Experimental design for quantifying single substrate degrada-tion rates. (A) JSOO6 LT cells were transformed with the single substrate pET28 plasmid were induced with 2 mM IPTG (indicated by the presence of red fluores-cence) for two hours. GFP fluorescence increased during this time. Then, the in-ducing medium was rapidly and accurately removed from the cells and replaced with non-inducing medium. (B) Fluorescence decay was measured over time.

M₁

M₂

W_B C

W

w

0 min 33 min 48 min 54 min 60 min

66 min 72 min 78 min 84 min 90 min

A B

C

Figure 4.4 : Microfluidic setup for single substrate degradation experiments.

(A) Bacterial DAW microfluidic design, where green indicates the cell trapping area, orange indicates the choatic mixers that thoroughly mix the ratios of media coming from the DAW (circled in red). M₁ and M₂ are media ports, W_B is a water balance for the DAW, C is the cell port, where cells are loaded into the device, and W_W is the waster port. (B) Appearance of the DAW at 0% inducing media, 50% inducing media, and 100% inducing media. (C) Images at 0 and 33 min of cells growing in 100% inducer (2 mM IPTG). Image at 48 min of the trapping regions, as media is being removed from the system. Images at 54 through 90 min showing cells growing in the absence of inducers; fluorescence decay is observed.

Figure 4.5 : General plasmid design for the dual feedback oscillator two-plasmid system. The repressor two-plasmid (pZA14LacI) has a p15A Amp^R backbone with the hybrid promoter p_lac/araregulating the expression of lacI-ssrA. The activator plasmid (pJS167 AraC-GFP) has a pBR322 Kan^Rbackbone with the hybrid promoter p_lac/ara regulating the expression of araC-ssrA and gfp-laa.

4.1.2 ssrA Degradation Variants for Dual Feedback Oscillator