Results

Two component system

Plasmid Engineering

After we transformed the Gibson Assembly ligation product into E. coli, we cultured the transformed bacteria on solid media with antibiotic Chloramphenicol(Cm). In order to confirm our ligation result, we picked several colonies and amplified them in liquid LB + Cm media to perform plasmid extraction. From Figure 1, we see that colony 16 had the desired complete plasmid.

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Figure 1. The result of enzyme digestion confirmation of the Gibson assembly results from different bacteria colonies. The numbers labeled above the wells are the assigned number of the bacteria colony. The bacteria of colony 16 carry the complete plasmid of pSB1C3-phla-mCerulean-p3-SaeRS plasmid.

We cultured bacteria from colony 16 in liquid media to double-confirm our result. From Figure 2b, we can be certain that we successfully constructed the pSB1C3-phla-mCerulean-p3-SaeRS plasmid and transformed it into E. coli.

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Figure 2a. The result of enzyme digestion confirmation of the colony 16 plasmid construction. The wells labeled “S” are the pSB1C3-phla-mCerulean-p3-SaeRS plasmid (Fig. 2b), while wells labeled “C” are the pSB1C3-J364007_Promoter_RBS_T1_T2_with_mCerulean_(hGFP)_CDS plasmid (Fig. 2c). The C groups serve as a comparative reference to ensure the functionality of the digestive enzymes.
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Figure 2b. The plasmid map of the pSB1C3-phla-mCerulean-p3-SaeRS plasmid.
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Figure 2c. The plasmid map of the pSB1C3-J364007_Promoter_RBS_T1_T2_with_mCerulean_(hGFP)_CDS plasmid.

To assemble the pSB1C3-phla-mCerulean-BBa_J23100-SaeRS plasmid, we are in the process of cloning three DNA fragments through PCR and subsequently ligating them using Gibson assembly. Currently, we have completed the PCR for one fragment, and the construction of the pSB1C3-phla-mCerulean-p3-Sae plasmid (BBa_K4822005+BBa_K4822007). Our experiment is still ongoing.

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Figure 3. The Sae_mut_f + Saeplasmid_r primer pair PCR product for J23100-Sae plasmid construction.

We extracted protein from the successfully transformed bacteria and performed SDS-PAGE. Due to our primer design, we only added one His tag after the SaeS protein in our plasmid construct. Since the genes for SaeR and SaeS are situated downstream of the same promoter and the two genes adjoin, confirming the expression of one protein is to ensure the expression of the gene saeRS. Therefore, we only performed protein purification and SDS-PAGE on the SaeS protein. From Figure 4, we can see that the bacteria expressed the saeRS gene and produced the protein.

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Figure 4. The result of SDS-PAGE with the SaeS protein.

Protein function

To investigate the sensitivity of our Sae plasmid device, we conducted fluorescent intensity and OD600 measurements to investigate the relationship between fluorescent density and Zn2+ concentration. We performed the Zn2+ test twice, eliminating data with OD600 measurements below 0.2 to mitigate significant errors caused by the limited bacterial numbers. We assume that the growth problem in high Zn2+ concentration groups is due to the high osmotic pressure. We noted that for Zn2+ concentrations below 1mM, the trend in fluorescent density is not distinct. However, within the range of 1 to 1.4mM Zn2+ concentration, there is a notable correlation between fluorescent density and Zn2+ concentration. However, our experiment only measured fluorescent density in Zn2+ concentrations up to 1.4 mM. We plan to conduct more experiments to collect more data to determine the sensitivity of our plasmid device.

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Figure 5. The result of the SaeRS device(BBa_K4822005+BBa_K4822007) Zn2+ sensitivity test.

L-Tyrosine

Plasmid Engineering

After obtaining the desired DNA fragments, two molar ratios of vector DNA and insert DNA were implemented, 1:1 and 1:3, respectively, to determine a better recipe for successfully ligating these two fragments.

Upon examining the results of transformation and culture, we observed that LB + Ampicillin agar plates with a 1:3 ratio had multiple colonies grown, whereas plates with a 1:1 ratio did not yield any colonies.

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Figure 6. Agar plate result of ligation using two different ratios. Plate on the left side was 1:3 ratio. Plate on the right side was 1:1 ratio.

After receiving the results of ligation, we proceeded to confirm the length of the constructed plasmid. We chose two restriction enzyme sites, BamHI and NdeI, which would generate distinct DNA fragment lengths that we could differentiate. Two colonies were selected randomly, each of which went through the aforementioned process.

The results were analyzed using agarose gel electrophoresis. Colony 1 and Colony 2 were divided into two groups and subjected to restriction enzyme digestion using BamHI and NdeI, respectively. After cutting with two BamHI sites, the DNA fragment lengths should be 5412 and 2493 base pairs. When cut with two NdeI sites, the DNA fragment lengths were 6052 and 1853 base pairs. The confirmation results indicated that both Colony 1 and Colony 2 contained the desired plasmid. Consequently, we proceeded with Colony 1 for the subsequent experiment.

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Figure 7. The electrophoresis results from restriction enzyme digestion products of two different colonies. C1 represents Colony 1 and C2 represents Colony 2.

Protein function

We measured the correlation between fluorescent density and Tyrosine concentration. Correlation coefficient R2 is 0.8542, which signifies notable correlation. This result shows that our TyrR device is applicable, but there is still room for improvement

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Figure 8. The plotting graph of the correlation between fluorescent density and Tyrosine concentration.

Extracellular Electron Transfer Pathway

pSB1C3-phla-(MtrCAB+tag)-Sae plasmid construction

After we received pETSXM2-pLacI-mtrCAB plasmid from Addgene, we conducted enzyme digestion to check the correctness of this plasmid. We use restriction enzymes XhoI and BamHI to digest the plasmid DNA. Only one cutting site enzyme digestion allowed us to check the total length of pETSXM2-pLacI-mtrCAB plasmid while several enzyme cutting site digestion allowed us to check if it is the correct plasmid DNA. By analyzing the gene map (Figure 9.), we predicted that the result of digestion by XhoI and BamHI would have two bands at 6.7k and 2.3k. Also, there is a chance that our pETSXM2-pLacI-mtrCAB plasmid hasn't been digested completely. So, the band at 9k still showed up. From the electrophoresis result (Figure 10.), we can learn that this plasmid DNA is correct.

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Figure 9. Plasmid map of the pETSXM2-pLacI-mtrCAB plasmid.
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Figure 10. The result of enzyme digestion confirmation of the pETSXM2-pLacI-mtrCAB plasmid.

Afterward, we kept extracting pETSXM2-pLacI-mtrCAB plasmid to obtain enough amount of plasmid to do the Polymerase Chain Reaction(PCR) to amplify mtrCAB fragments and add on two cutting sites, SacII and BamHI.

Then, we use SacII and BamHI to digest both vector DNA, pSB1C3-phla-Sae plasmid, and mtrCAB fragment. By running electrophoresis (Figure 11.), we can use gel extraction to obtain purified DNA fragments. After that, we did ligation to make them a complete plasmid. Lastly, we transformed the modified plasmid into E. coli DH5α. We cultured the bacteria on the LB agar plate with chloramphenicol as a selection marker.

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Figure 11. The result of enzyme digestion confirmation of the mtrCAB PCR product and the vector DNA.

After the bacteria survived on the LB agar plate with chloramphenicol. We have to check whether the bacteria is harboring the correct plasmid. We extracted plasmid DNA from it and digested the plasmid DNA with a restriction enzyme, XbaI. From the gene map, it is clear that we can obtain 3 fragments after digesting by the restriction enzyme XbaI. The size of each fragment will be about 5k, 3k, 1.5k. The electrophoresis results of colony 1 and colony 3 can fit in the prediction and prove our plasmid construction successfully. (Figure 12.)

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Figure 12. The result of enzyme digestion confirmation of the plasmid DNA extracted from transformation strain. (U: Uncut, C: Cut by XbaI)

pYJ_BBa_J23100_CcmAH_BBa_B0015 plasmid

The ccmA-H gene cluster is taken from the E. coli genome. We choose BBa_J23100 and BBa_B0034 as promoter and RBS (p-r), and BBa_B0015 as terminator. Moreover, we picked replication origin and Ampicillin resistance gene (O-A) from pYJ-phla-LacZ to make the engineered bacteria selectable with LB+Chloramphenicol+Ampicillin medium. In these fragments, ccmA-H, p-r, and O-A are prepared by PCR, and BBa_B0015 is prepared from pSB1C3-mCerulean by enzyme digestion. One problem we faced is that p-r fragments are too short to stain clearly under UV light. Therefore, we prepared more than 30 times p-r PCR product volume, then concentrated 30X to see the band while cutting the gel. Another problem we faced is the ligation of this plasmid. Because running out of time, we are not able to finish this experiment before the wiki freezes. We tried several rates of concentration, but could not get the right plasmid before we ran out of PCR products and polymerase.

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Figure 13. The result of electrophoresis of our PCR product.
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Figure 6. Plasmid map of pYJ-BBa_J23100-CcmAH-BBa_B0015

Project Achievements

What we did achieve

  1. Construction of sae device to detect Zn2+, and report by mCerulean fluorescent density.
  2. Construction of the Tyr device to detect Tyrosine, and report by mRFP1 fluorescent density.
  3. Design two plasmids for EET pathway construction.

What we didn’t achieve

  1. Construction of BBa_J23100-saeRS device.
  2. Extraction of Streptomyces avermitilis gnomic DNA.
  3. Construction of pYJ_BBa_J23100_CcmAH_BBa_B0015 plasmid.
  4. Co-transformation of pSB1C3-phla-(MtrCAB+tag)-Sae plasmid and pYJ_BBa_J23100_CcmAH_BBa_B0015 plasmid into the E. coli BL21.
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Contact

National Yang Ming Chiao Tung University Yangming Campus
No. 155, Section 2, Linong St, Beitou District, Taipei City, 112

+886-2-2826-7000
nycu.taipei.igem@gmail.com

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