Characterization

Overview

To test the functionality of the lacI regulated promoter used in our BioBricks designed for its expression in the E. coli SHuffle strain, our team characterized the following parts:

BBa_R0010 Promoter (lacI regulated)
BBa_J04450 RFP Coding Device
BBa_K875009 LL 37 - Cathelicidin

Introduction

Since two of the peptides involved in our project, PsDef1 and WAMP1b, contain multiple disulfide bonds, proper protein folding is required. Different approaches were considered to achieve this, and the principal one was looking for a proper promoter for our BioBricks. The usage of inducible promoters is ideal to avoid unwanted characteristics in recombinant proteins, such as inclusion bodies.¹ It has been shown that regulated promoters yield higher quantities of soluble proteins.² Thus, we designed our composite parts for these two peptides under the regulation of part BBa_R0010, a lacI regulated promoter inducible by IPTG.

Another aspect we considered was choosing the ideal expression chassis. E. coli SHuffle T7 Express®️ form New England BioLabs®️ was chosen for its ability to produce disulfide bonded proteins in its cytoplasm. However, no information was found in the Registry about the usage of part BBa_R0010 in SHuffle strains. In order to test this lacI regulated promoter in our chassis, we transformed part BBa_J04450, which expresses RFP under this promoter, into E. coli SHuffle T7 Express®️.

We successfully characterized parts BBa_R0010 and BBa_J04450. RFP expression was induced in different temperature conditions and IPTG concentration to determine the ideal conditions for the proper functionality of the promoter. Protein expression was analysed through an SDS-PAGE. Through this characterization, we have added information to the Registry for future teams who wish to use SHuffle®️ strains in their projects.

Experimentation

First, 3 μL of the 10 pg/μL vial of Part BBa_J04450 (RFP construct) from the Competent Cell Kit were transformed into chemically competent E. coli SHuffle T7 Express®️ cells. Transformed cells were grown on LB agar with chloramphenicol, for BBa_J04450 (RFP construct) was located in the pSB1C3 backbone. Small, rounded, red colonies grew on the surface of the agar.

Figure 1. E. coli SHuffle T7 Express®️ transformed with BBa_J04450 grown on LB Agar.

Once the expression chassis was transformed, protein expression was induced. To do so, two tubes of 10 mL of liquid culture of untransformed E. coli SHuffle T7 Express®️ and six of SHuffle cells transformed with BBa_J04450 were prepared. Once the tubes reached an OD₆₀₀ of 0.4, they were incubated for 5 hours under the conditions of temperature and IPTG concentration shown in the following table.

Table 1. Conditions of induction for characterization of production of RFP.

Well	37°C	Well	30°C
Negative control	Untransformed control	Negative control	Untransformed control
1	Uninduced control (without IPTG)	4	Uninduced control (without IPTG)
2	Transformed cells, 0.2 mM IPTG	5	Transformed cells, 0.2 mM IPTG
3	Transformed cells, 0.4 mM IPTG	6	Transformed cells, 0.4 mM IPTG

Induction temperatures were chosen based on commonly used conditions (37°C) and recommendations of the supplier (30°C). The uninduced control consists of transformed cells without IPTG which meant to serve as an indicator of leakiness in the promoter. IPTG concentrations were chosen based on conditions that had worked previously for us when using a T7 promoter (0.2 mM) and recommendations of the supplier (0.4 mM).

After 5 hours of incubation at 225 rpm, cells were centrifuged at 5000 rpm and 4°C for 7 minutes. The pellet was resuspended in a lysis solution with lysozyme and cells were lysed through multiple freeze/thaw cycles. The cell lysates were centrifuged at 21,380 g to separate the soluble protein fraction (supernatant) which was retrieved for its evaluation and quantification. Then, 10 μL of each sample with 50 μg of total protein were mixed with 10 μL of SDS loading buffer and loaded into a 12% polyacrylamide gel. The gel was ran at 10 mA until the running front entered the separating gel; then the current was increased to 15 mA.

Results

The following image shows the results obtained from the SDS-PAGE.

Figure 2. SDS-PAGE (12%) of E. coli SHuffle T7 Express®️ transformed with BBa_J04450 (RFP construct).(Negative control:E.coli Shuffle untransformed cells at 37°C, 1.RFP transformed cells without induction, 2.RFP transformed cells with 0.2mM of IPTG at 37°C, 3.RFP transformed cells with 0.4mM of IPTG at 37°C, Negative control: E.coli Shuffle untransformed cells at 30°C, 4.RFP transformed cells without induction, 5.RFP transformed cells with 0.2mM of IPTG at 30°C, 6.RFP transformed cells with 0.4mM of IPTG at 30°C) See Table 1.

Two prominent bands can be appreciated in the last two columns, corresponding to induction conditions of 0.2 and 0.4 mM IPTG at 30°C, close to the 25 kDa band of the Precision Plus Protein^TM Dual Xtra Prestained Protein Standards. It was concluded that they correspond to RFP, a 26 kDa protein.

When taking a closer look, the band corresponding to RFP can be seen in each column with exception of the untransformed cells controls (negative controls). The uninduced controls (column 1 for 37°C and column 4 for 30°C) without IPTG were expected to show no bands if the promoter lacked leakiness; nevertheless, a faint band can be seen for both controls. Bands corresponding to 0.2 and 0.4 mM IPTG for both temperatures (columns 2 and 3 for 37°C and columns 5 and 6 for 30°C) show stronger bands compared to the uninduced control. Thus, the promoter is slightly leaky, but the addition of proper amounts of IPTG increases protein production. When comparing temperatures, incubation at 30°C clearly yields a higher amount of RFP, but both concentrations yield similar amounts of expression.

Conclusions

Our team has added relevant information to the Registry for future teams who might want to use a SHuffle strain in their projects. We have demonstrated the functionality of promoter BBa_R0010 in E. coli SHuffle T7 Express®️ as well as ideal conditions for protein production by characterizing part BBa_J04450, an RFP construct. E. coli SHuffle T7 Express®️ produces higher amounts of protein at 30°C, and concentrations of 0.2 or 0.4 mM IPTG are enough to induce expression when using this lacI regulated promoter.

LL 37

We realized that the few characterizations of LL37 were all qualitative. This is why Tec-Chihuahua team undertook the task of characterizing this part in order to report a quantitative absorbance study. Our team worked on another characterization; this time for part BBa_K875009. This is a basic part that codes for LL 37, a human peptide with antimicrobial activity against Gram-positive and Gram-negative bacteria.³ Some bacteria that are susceptible to this peptide include E. coli, P. aeruginosa, and S. aureus.⁴ Being one of the most used organisms for recombinant protein expression, we aimed to evaluate the effects that LL 37 may have on the growth of Escherichia coli as an expression chassis.

Expression of toxic proteins may adversely affect the viability of the chassis. In order to test E. coli as a viable host for LL 37 production, E. coli BL21 (DE3) cells were transformed with part BBa_K2959000, which is a composite of our creation capable of expressing LL 37 under the regulation of the T7 promoter. Three flasks with 30 mL of LB broth were inoculated with 3 mL of an overnight culture of transformed cells. Another flask with 30 mL of LB broth was inoculated with 3 mL of untransformed cells to use as a control. The four flasks were incubated at 37°C and 225 rpm until OD₆₀₀ reached 0.8. The flasks with transformed cells were treated with different concentrations of IPTG (0.1, 0.5, and 1 mM) to induce protein production. Then, optical density was measured every hour for five hours to measure growth. Results are presented in table 2 and figure 3.

Table 2. OD₆₀₀ of transformed and untransformed E. coli BL21 (DE3) cultures treated with different concentrations of IPTG.

	0 h	1 h	2 h	3 h	4 h	5 h
Untransformed BL21 (DE3)	0.859	1.238	1.511	1.636	1.709	1.752
Transformed BL21 (DE3), 0.1 mM IPTG	0.784	1.238	1.362	1.451	1.549	1.6
Transformed BL21 (DE3), 0.5 mM IPTG	0.80	1.218	1.346	1.418	1.51	1.573
Transformed BL21 (DE3), 1.0 mM IPTG	0.81	1.197	1.297	1.351	1.427	1.481

The results obtained (Figure 3) show a distinct difference in absorbance, which correlates to culture growth, between untransformed and transformed E. coli. After 5 hours of incubation, untransformed BL21 (DE3) (blue line) indicated an OD600 of 1.752. Previous data, compared to transformed BL21 (DE3) with LL37 (yellow line) with the highest induction condition and time (1mM of IPTG and 5 hours), shows an OD600 of 1.481 which denotes a reduction in growth of approximately 15.5%. Untransformed cells grew better, indicating that LL37 production has a negative effect on bacterial growth. Concentration of IPTG and absorbance showed an inversely proportional relationship. Higher concentrations of IPTG induce a larger production of LL 37 and, due to its toxicity towards its host, it may have caused this partial inhibition. Thus, expression of LL 37 reduces bacterial growth; however, no significant reduction in total growth was perceived. In conclusion, growth inhibition is not significant enough to consider the expression of LL 37 in E. coli BL21 (DE3) non-viable.

Figure 3. Absorbance curve measured at 600 nm for transformed and untransformed E. coli BL21 (DE3) cultures treated with different concentrations of IPTG.

References

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