Team:NAU-CHINA/Results

NAU-iGEM

Overview

Our results are about proving the feasibility of the anti-Plasmodium system. We first tested the HlyA secretion system in E.coli DH5α but failed, then we started to verify the expression and cleavage effect of the plasmid in E.coli. After that we succeeded in electroporation, thus we improved our project in the replace of promoters in S. marcescens. The following are our specific experimental results.

Effect of HlyA secretion system in E.coli

BBa_K3139001 UTR: http://parts.igem.org/wiki/index.php?title=Part:BBa_K3139001
BBa_K528004 RBS measurement part: http://parts.igem.org/Part:BBa_K528004:
BBa_K3139017 RBS measurement part with UTR (improved): http://parts.igem.org/wiki/index.php?title=Part:BBa_K3139017
BBa_K1166002 HlyA secretion system: http://parts.igem.org/Part:BBa_K1166002

Purpose

We constructed the RFP-HlyA plasmid and removed the inducible promoter and the double terminator in the HlyA secretion system to construct the simplified RFP-HlyA plasmid. Then, we verified the secretion effects of the two systems.

Pre-experiment

Every 1h, a set of samples were placed in the shaker until all samples were placed. The OD₆₀₀ and fluorescence value of bacterial fluid, the supernatant after centrifugation, and the bacterial liquid resuspended in 1×PBS after removing the supernatant were measured. The results showed that neither the HlyA secretion system nor the simplified HlyA secretion system worked properly under 37℃ and 28℃, since the bacteria didn’t seem to show any fluorescence at 36 h. So we canceled this experiment, and decided to design an “enhancer” in our HlyA secretion system to debug, and repeat the experiments for further validations. For further validation, we improved BBa_K528004 RBS measurement part. To improve the stability of mRNA and ribosome binding probability, we inserted a sequence at the 5'-UTR of the RFP coding sequence of the RFP-HlyA plasmid. Then, we induced the DH5α strain with 0.2% L-arabinose. The bacterial fluid, the supernatant after centrifugation, and the bacterial liquid resuspended in 1×RBS after removing the supernatant were taken, and the OD₆₀₀ and fluorescence values were measured. The results showed that the re-improved HlyA secretion system also did not meet the expected extracellular secretion effect (Fig. 1.). But the expression of RFP was improved greatly.

Note: The negative values shown in this figure are caused by the error of the background, but it does not affect the relations of the fluorescence values at different times of each treatment.

Note: This figure shows the total fluorescence intensity of the bacterial fluid.

Note: -ara: no 0.2% arabinose induction
+ara: induction culture with 0.2% arabinose for 16 hours
-utr: no BBa K3139001 insertion
+utr: BBa K3139001 insertion

We verified the BBa_K1166002 again, finding the secretion effect is not significant. The BBa_K528004 RBS measurement part inserted UTR had improved its function significantly. However, the secretion effect decreased after L-arabinose induction. And the total amount of fluorescence also decreased with the use of L-arabinose. At the same time, we measured that the numbers of bacteria in different treatments, which were approximately at the same level.

BBa_K3139001 UTR
Discussion：

After two experiments, we believe that the function of BBa_K1166002 HlyA secretion system cannot be observed under our experimental conditions. We have proposed the following possibilities and these may be verified in future research.

1.How to explain the abnormal function of BBa_K1166002 HlyA secretion system?

According to some existing reports, the transmembrane complex of the secretory system also has a TolC protein, but its coding gene is not presented in the part. So, the function of the transmembrane complex that is encoded by the part may be missing, and the RFP they express will be retained in the intracellular and periplasmic spaces. Therefore, we cannot observe the realization of its function.

2.How to explain the extracellular fluorescence?

Since the number of bacteria is kept at the same level, we can roughly rule out cell lysis due to protein accumulation. We hypothesized that there is another mode of transportation in E. coli DH5α that can transport excessive fluorescent protein to the extracellular domain, and this transport mechanism will work faster with the increase in the number of fluorescent proteins.

3.Why did the L-arabinose induction reduce the total fluorescence?

L-arabinose induced the expression of hlyB and hlyD in BBa_K1166002, which are functionless, and even a waste of cell’s energy, since the effective transmembrane complex is not available. So the resources that should be given to the expression of rfp reduce, leading the decrease of its expression and fluorescece intensity.

4.How to explain the phenomenon described in 2 is more obvious after inserting UTR?

L-arabinose induced the expression of hlyB and hlyD in BBa_K1166002, which are functionless, and even a waste of cell’s energy, since the effective transmembrane complex is not available. So the resources that should be given to the expression of rfp reduce, leading the decrease of its expression and fluorescece intensity.

Verification of the expression and cleavage of the plasmid in E.coli

The ideal strain that we hoped to use is Serratia marcescens. However, since we did not know the conditions of the electroporation of S. marcescens at the beginning, we prepared the experimental materials both of E. coli and S. marcescens in parallel.

1.We transferred the plasmid (Fig. 1) into BL21 strain and cultured it in LB medium to obtain supernatant after ultrasonication. Then we purified our target His tagged protein by using Ni-NTA magnetic beads. It was predicted that TEVp cleavage would not be so complete that nine different sizes of protein bands and TEVp's own band would appear. In the preliminary experiment, we electrophoresed the purified protein on sodium dodecyl sulfate (SDS) -12.5% (wt/vol) polyacrylamide gel, followed by Western Blot to verify the expression and cleavage effect of this plasmid in the bacteria. We use anti-His antibody as primary antibody. In the result, the band of anti-Plasmodium effector fusion protein (75.1 kDa) in the sample without TEVp is darker than the one in the sample with TEVp ( Fig. 2).

We also purified our target His tagged protein by using Ni-NTA magnetic beads from supernatant. And we got proteins sizing under 10 kDa by ultrafiltration device to conduct the mass spectrometry analysis. We confirmed the existence of the smallest monomer protein (7.2 kDa) produced by TEVp completely cleavage (Fig .3). Above two experiments strongly prove that our plasmid could express and cleave to produce anti-Plasmodium peptides.

2. Then we did formal experiments. We continued to conduct WB for several times. Comparing to the results of fresh bacteria liquid and the results of bacteria liquid stored at 4℃ for 3 days, we surprisingly found that : both samples had the obvious band of TEVp (29.5 kDa). However, the band of large protein existed only in the fresh sample, not in the sample stored for 3 days, while the smallest monomer protein (7.2 kDa) produced by TEVp completely cleavage exited only in the sample stored for 3 days, which suggested that the secondary cutting was more complete than the first one (Fig. 4). This result proved that TEVp was still able to perform the cutting function efficiently even at 4 ℃.

EFFECT OF HasA SECRETION SYSTEM IN S. marcescence

To verify that the HasA system can successfully secrete proteins in Serratia, we constructed the RFP-HasA plasmid.

We have conducted a long-term exploration of the electroporation conditions of Serratia, and finally found that the electroporation voltage of Serratia JCM11315 was 2100v. Afterwards, we transferred the plasmid into the Serratia JCM11315 by electroporation.

However, we were unable to screen out the correct strain with ampicillin. After verification, we found that Serratia itself had ampicillin resistance before electroporation, so we changed the plasmid skeleton. We used chloramphenicol to screen positive strains.

To verify the actual effect of HasA secretion over time, we cultured positive strains overnight and inoculated some of them into a new liquid medium for a total of 72 hours on a four-hour gradient.

The protein concentration of the bacterial supernatant at different times was determined by a microplate reader. We obtained satisfactory data and successfully verified the secretion effect of HasA, which means our system can work in Serratia.

Promoter replacement in S. marcescens

Since we found out the electroporation condition of S. marcescens. Then we carried out related experiment and verified that HasA secretion system can work well in S. marcescens. The expression ratio of TEVp and anti-Plasmodium effector fusion protein can influence the effect of cleavage and the concentration of effectors. To obtain the best cleavage efficiency, we planned to replace the promoter of TEVp, compared the cleavage efficiency, and finally chose the most suitable promoter. We selected three promoters: J23111，J23110 and J23106, from the family of constitutive promoter parts, to construct 3 plasmids (Fig. 5).

Fig. 5 Plasmids in S. marcescens

We transformed these three plasmids into S. marcescens 11315, and cultured them in LB medium for 60h to obtain supernatants after centrifugation. Then we purified our target His tagged protein by using Ni-NTA magnetic beads. The WB result shows several bands. We defined the smallest monomer protein band (7.2 kDa) which was produced by completely cutting as the representative of cutting effect, and the band of TEVp (29.5 kDa) represents the promoter strength of TEVp. From the grayscale of the TEVp and monomer protein bands (Table. 1), we found out the cutting effect of the strongest promoter J23111 was weak than the cutting effect promoter J23106, which has the best cutting effect. (Fig. 6) It also supported the results of our model that changing the strength of promoters can change the cutting effect and the concentration of single effectors in extracellular, thus providing theoretical support for the practical application of our project.

	J23110	J23111	J23106
His-TEVp	48142	131106	68468
His-ADdLP	19234	54615	71026

Table. 1 the total grayscale of His-TEVp and monomer protein bands

Characterization

Contribution
Summary: We measured the fluorescence signal of mRFP in buffer under different pH values and temperatures. Report genes are essential for every team to construct genetic pathways. We believe that every team has a J04450 complex. After all, it is our favorite report gene!
This year, the NAU iGEM characterized E1010. Based on the work of the previous team work, we explored the tolerance of E1010 expression products at different pH values and temperatures.Pre-experiment[edit]We dispensed a small amount of the sample into 12 PCR tubes, and a gradient test at 65-95℃ was conducted in a PCR machine for 10 minutes.
We found that the protein samples showed significant denaturation when the temperature was higher than 75℃. So, we set the experimental temperature later at and above 75℃.
The purified sample was dispensed into four PCR tubes and the pH was） adjusted to 4.0, 8.0, 10.28, respectively. Under each pH value, we dispensed each sample into 12 PCR tubes（50uL sample + 50uL buffer）, and a gradient test at 75-95℃ was conducted in a PCR machine for 10 minutes.
We measured the fluorescence intensity of the product under the microplate reader (excitation wavelength was 584 nm, and absorption wavelength was 611 nm).

Analysis: The results show that the product of J04450 begins to show significant denaturation at 84.4℃, and the fluorescence value of the protein no longer changes significantly at the temperature of around 92.5℃, which means E1010 product is completely denatured (shown in our characterization map with a small amount of protein precipitation and the color is pale yellow).
The temperature (from 75.4℃ to 90.3℃) and fluorescence values show a significant linear relationship .
At the same temperature, the pH=4.0 and pH=8.0 curves were basically consistent. The stability of the E1010 expression products at these two pH values was significantly better than that under the condition of pH=10.28.Discussion[edit]Report genes are required for the construction of most gene pathways. In the future iGEM journey, other teams and we may use some bacteria, like thermophiles, that grow in extreme conditions. We hope we can still use our familiar report gene in these bacteria to detect protein secretion and other related circumstances, so we conducted these tests.
We simulated the extreme environments in which we released the expression products to the extracellular—acid, weak acid, alkaline, and high temperatures—by adjusting the temperature gradients and pH values.
We hope that our experimental results can provide other teams with a reference for the stability of report gene expression products in extreme environments.

References

[1]Sakural N, Komatsubara S. Simple and versatile electrotransformation of Serratia marcescens Sr41[J]. Letters in Applied Microbiology, 2010, 23(1):23-26.
[2]Sapriel G , Wandersman C , Delepelaire P . The N Terminus of the HasA Protein and the SecB Chaperone Cooperate in the Efficient Targeting and Secretion of HasA via the ATP-binding Cassette Transporter[J]. Journal of Biological Chemistry, 2002, 277(8):6726-6732.
[3]S Létoffé, Ghigo J M , Wandersman C . Secretion of the Serratia marcescens HasA protein by an ABC transporter.[J]. Journal of Bacteriology, 1994, 176(17):5372-7.
[4]Sapriel G , Wandersman C , Delepelaire P . The SecB Chaperone Is Bifunctional in Serratia marcescens: SecB Is Involved in the Sec Pathway and Required for HasA Secretion by the ABC Transporter[J]. Journal of Bacteriology, 2003, 185(1):80-88.