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Insertion of the Metallothionein Cup-1-1 domain into Bacillus subtilis flagellin

Organisms

Hag from Bacillus subtilis and Cup1-1 from Saccharomyces cerevisiae.

Cloning procedure

Cup1-1 was cloned into B. subtilis flagellin via Gibson assembly to create the fusion protein Hag-Cup1-1. This construct was assembled in pET24d for overexpression of the protein, and the resulting Hag-Cup1-1 construct was then transferred to pMAD for the integration into the genome of B. subtilis. The final construct was additionally cloned into the iGEM backbone pSB1C3 for submission to the registry.

Expression test

In order to overexpress Hag-Cup1-1 an expression test was performed. Therefore piGEM21 was transformed into E. coli BL21(DE3). To prevent the monomeric protein from assembling into polymeric filaments a vector containing a chaperone (FliS) was co-transformed. For the expression test a 20-mL culture was grown to an OD of 0.7. Pre-induction (PI) and induction (I) samples were taken from the culture and prepared for gel analysis. After centrifuging the samples, the pellet was resuspended in water and SDS buffer and heated to 95°C. Following an additional centrifugation step, 5 and 10 µL, respectively, were analyzed on an SDS-PAGE gel.

Gel analysis indicates that Hag-Cup1-1 was successfully expressed in E. coli since a high concentration of a specific protein could be seen after induction.

Overexpression

Since the expression test was successful, protein expression was scaled up. For that purpose, 2 x 1 L LB with the appropriate antibiotic was inoculated with every clone on the co-transformation plate. Protein expression was induced through addition of lactose (12,5 g/L), and the cells were cultured overnight (ON) at 30 °C.

According to the SDS-PAGE gel, the protein of interest (Hag-Cup1-1) is present in a high amount and can be used for crystallization after purification. Hag (WT flagellin) served as a control for the overproduction of flagellin.

Purification and Crystallization of Hag-Cup1-1

Cells were lysed with a microfluidizer and the protein was purified from the supernatant.

After the Ni-NTA purification step, the fractions containing protein were pooled and concentrated in on a centrifugal filter. Next, the protein was injected onto a gel filtration column.

The Load, Flow-through, Wash and Elution samples 1-6 from the Ni-NTA purification were analyzed on an SDS-PAGE gel to confirm the presence of Hag-Cup1-1 in the elution fractions.

Gel filtration: 40 µL of different fractions (C7, C9, C11, D1, D3, D5, D7) were collected from the elution peak and analyzed via SDS-PAGE.

The gel analysis reveals that the elution fractions contain the protein in a high amount. Elution fractions C7-D7 were pooled and concentrated to a volume of 200 µL. The concentrated protein was screened for crystals in an undiluted and 1:2 (protein:buffer) diluted state with JCSG Core I-IV solutions.

Motility assays B. subtilis with Hag-Cup1-1

For testing the motility of B. subtilis strains with the metallothionein inserted into their flagellum, swimming and swarming assays were performed.

Swarming agar (0,7% LB-Agar) and swimming agar (0,3% LB-Agar) plates were prepared. A defined amount of cells was spotted in the middle of these plates and incubated at 37 °C. The swimming and swarming behavior of wildtype strains and the Hag-Cup1-1 strain (in duplicate) was documented for 2.5 hours. After every time point, the progress of B. subtilis motility was marked with a line. Evaluation of the swimming and swarming assays lead to the conclusion that the B. subtilis strain with Cup1-1 inserted into the flagellin is no longer able to swim or swarm. This could be due to multiple reasons that have to be examined in further experiments.

Summary of the motility of the Bacillus strain with Hag-Cup1-1:

Expression of Flagellin

In order to analyze the expression of the flagellum in the Bacillus subtilis mutant, cultures were grown to an OD of 0.6-0.8. Cells were spun down and prepared for analysis on an SDS-PAGE gel. Flagellin produced in E. coli was used as a control. In the case of positive expression, analyzed culture samples are expected to migrate as far as the E. coli produced samples.

The mutant with the inserted Cup showed no significant expression of flagellin. Export of flagellin may not be possible for Bacillus subtilis because the size/folding prevent the proper production and assembly of the proteins into a functioning flagellum. Protein degradation or limited expression may account for the absent band on the SDS-PAGE gel. These observations agree with the previously performed swarming assays.

Plan B

As Bacillus strains carrying the functionalized flagellin (Hag-Cup1-1) are not able to swim and swarm and do also not express flagellin, we developed different ideas to solve this challenge. The best idea seemed to be the insertion of the D2 domain of the Salmonella typhimurium flagellin as a linker to make the filament more flexible. A gene fragment encoding this construct was synthesized by Integrated DNA Technologies.

Cloning procedure

The cloning procedure for the altered Hag-D2-Cup construct started with PCR amplification of Hag-D2-Cup from the synthesized gene fragment. This fragment was then assembled into the cloning vector pMAD and the expression vector pET24d.

Expression test

Before overexpressing Hag-D2-Cup, an expression test was performed. Bacillus cultures were grown to an optical density of 0.6-0.8. Samples were collected, spun down and prepared for analysis on an SDS-PAGE gel. It could be observed the Bacillus strain with Hag-D2-Cup does not express the modified flagellin. For this reason, an overexpression was not performed and motility assays were planned instead.

Motility assays

To test the motility of B. subtilis strains with Hag-D2-Cup inserted into their flagellum, swimming and swarming assays were performed as previously described for Hag-Cup1-1. Swarming agar (0,7% LB-Agar) and swimming agar (0,3% LB-Agar) plates were prepared. A defined amount of cells was spotted in the middle of these plates and incubated at 37 °C. The swimming and swarming behavior of wildtype strains and the Hag-D2-Cup strain was documented for 5.5 h. At each time point, the progress of B. subtilis motility was marked with a line.

Evaluation of the swimming and swarming assays lead to the conclusion that the B. subtilis strain with Hag-D2-Cup inserted into the flagellin is no longer able to swim or swarm.

Hag-D2-Cup did not swarm or swim, which agrees with the results observed in the SDS-PAGE gel. Electron microscopy should bring insight into questions that arise from the previous experiments. In order to obtain more reliable results, the motility assays will have to be repeated.

Duplicate of motility assays B. subtilis with Hag-D2-Cup

Evaluation of the second motility assay leads to the same result as the previous assay. Therefore, electron microscopy was planned in order to detect whether the modified flagellin can assemble into a functioning flagellum.

Electron microscopy

Bacillus subtilis WT3619 and 3610 and D2-Cup strains were cultured in LB medium overnight in order to analyze the flagella in different states of growth. The cultures were incubated with shaking at 37 °C until they reached the exponential phase with an OD600 of 0.7 - 0.8, and until the cells reached the stationary phase. The cells were kept at the same OD by storage on ice until further processing for electron microscopy. Electron microscopy was performed by Dr. Thomas Heimerl (AG Meier, biology department).

Sequence and Features