Our aim in this project is to make a Colorectal Cancer bio-detection using aeBlue chromoprotein BioBrick plasmid backbone with an annealing site for iGEM standard primer, VF2 and VR. In achieving the said aim, we designed the LIRA OR Gate System biodevice sensitive to miR-21 and miR-92a that was then assembled into Escherichia coli Nissle 1917 (EcN). The LIRA OR Gate System was designed to include blue Chromoprotein thus producing blue color in feces when colorectal cancer is detected by the EcN colonies. Additionally, to characterize the aeBlue expression in the presence of miR-21 and miR-92a, we designed inserts that constitutively express miR-21 (Fig. 1a) and miR-92a (Fig. 1b). We plan to assemble these insert and transform them both into EcN cells along with the LIRA OR Gate plasmid to characterize its regulatory activity internally. The construct of LIRA OR Gate, miR-21, and miR-92a are shown in Figure 1. The working mechanism of LIRA OR Gate, miR-21, and miR-92a is shown in Figure 2.
Figure 1. (A) Design of LIRA OR Gate, (B) miRNA92a, and (C) miRNA21
Figure 2. Mechanism of LIRA OR Gate System and miR21, miR92a worked (Modified from Ma et al. 2022)
We succeeded in transforming aeBlue Chromoprotein plasmid into EcN, and the colonies are shown in Figure 3. After that, we tested the aeBlue plasmid stability inside the EcN, growth activity, and toxicity of EcN-aeBlue. The stability assay result shows that EcN-aeBlue can express chromoprotein up to 4 cycle of repetitions (Table 1). For the growth activity, modified EcN absorbance is measured by spectrophotometer in every 30 minutes until 16 hours. Growth curves for both bacteria cultures are shown in Figure 4. Generation time is calculated from the growth data displayed in Table 2 using the equation shown below that. Lastly, the toxicity assay of EcN-aeBlue shows that there is no significant difference in viability in modified EcN and control (EcN). Therefore, both EcN-aeBlue and EcN are nontoxic for colorectal cancer cell lines, as shown in Table 3.
Table 1. Plasmid Stability Assay of EcN-aeBlue interpretation
++ = strong colour in blue
+ = weak colour in blue
- = no blue color
Figure 3. EcN- aeBlue colonies
Figure 4. Growth rate curve of EcN-aeBlue and EcN
Table 2. Exponential growth of EcN-aeBlue and EcN
B: the number of bacteria counted in the beginning of interval time
b:the number of bacteria counted in the end of interval time
T: time
Here are the equations of bacterial growth rate
Equation of Bacteria Generation Time (G)
G=603.3log(bB)G = \frac{60}{3.3 \cdot \log\left(\frac{b}{B}\right)}
Equation of EcN-aeBlue Generation Time (G EcN-aeBlue)
GEcN-aeBlue=603.3log(0.47620.056)=19.56G_{\text{EcN-aeBlue}} = \frac{60}{3.3 \cdot \log\left(\frac{0.4762}{0.056}\right)} = 19.56
Equation of EcN-aeBlue Generation Time (G EcN-aeBlue)
GEcN-aeBlue=603.3log(0.4710.0631)=20.83G_{\text{EcN-aeBlue}} = \frac{60}{3.3 \cdot \log\left(\frac{0.471}{0.0631}\right)} = 20.83
G: generation time (minute)
B: the number of bacteria counted in the beginning of interval time (CFU/ml)
B: the number of bacteria counted in the end of interval time (CFU/ml)
T: interval time (minute)
Equation of Ecn-aeBlue growth relative rate with EcN
G Ecn-aeBlue: generation time of EcN-aeBlue (minute)
G EcN : generation time of EcN (minute)
Table 3. Toxicity Assay of EcN-aeBlue and EcN in colorectal cancer cell line
In the next step, we constructed the LIRA System, miR-21, and miR-92a. Since the limited time and the complexity of our project, we just focused on the engineered of LIRA OR Gate System assembled in a plasmid and transformed it into Escherichia coli Nissle 1917.
1. Gene construction
To construct our biodevide, we included several biobrick parts. The list of Biobrick parts can be seen on the Part Page.
Figure 5. Plasmid Design of the BioBrick
While constructing the plasmid with BioBrick parts, we isolated the EcN, shows in Figure 5, and confirmed by PCR Colony using primer Muta5 as forward and Muta6 as reverse for pMUT1 and primer Muta7 as forward and Muta8 as reverse for pMUT2 which shows in Table 3. pMUT1 and pMUT2 are the endogenous plasmid of EcN. The results of PCR Colony are shown in Figure 6 as pMUT1 confirmation and Figure 7 as pMUT2 confirmation.
Table 4. The sequence of Muta5, Muta6, Muta7, and Muta8 primers.
Figure 6. EcN colonies
Figure 7. pMUT1 Confirmation
Figure 8. pMUT2 Confirmation
We isolated those endogenous plasmid of EcN with ZymoPURE II Plasmid Kits and confirmed by amplifying it with muta5 and muta6 primer. The electrophoresis result is shown in Figure 9 and the DNA Concentration is quantified by Nanodrop. The result is shown in Table 5.
Figure 9. pMUT1 Isolation
TubeDNA Concentration (ng/uL)
Table 5. DNA Concentration of isolated pMUT1
For preparing the backbone, the confirmed pMUT1 were amplified by primer consisted of BioBrick part and Gibson assembly sequences as backbone. The products were confirmed at around 3216 bp shows in Figure 10. After that, the PCR products was purified by PCR Clean Up and the DNA Concentration was quantified by Nanodrop, shows in Table 6.
Figure 10. Amplification of pMUT1 Gibson as backbone
TubeDNA Concentration (ng/uL)
Table 6. DNA Concentration of pMUT1 Purified Product
For preparing the insert, we designed and synthesized LIRA OR Gate System (Twist Bioscience) with the sequence shows in Table 7. It was amplified by specifically designed primer with overhang Gibson assembly sequences for LIRA and resulted in Figure 11. It was purified by Zymo Gel Purification Kit and quantified by Nanodrop, resulted in Table 8.
Table 7. LIRA Sequences
Figure 11. Amplification of LIRA
TubeDNA Concentration (ng/uL)
Table 8. DNA Concentration of LIRA Purified Product
2. Assembly (using Gibson Assembly)
The purified backbone and insert that already had overhang sites were assembled using Gibson Assembly. The reaction mix are incubated in 50c for 1 hour in a thermal cycler.
3. Genetic Transformation
We transformed the assembled product to EcN competent cells that was made before using standard heat shock transformation protocol. As control, the transformation used pUC19a and NEBuilder also. After an overnight incubation, we selected randomly the colonies in one plate to confirm in PCR Colony using LIRA specific primer. The transformed colonies are shown in Figure 12. The result is shown in Figure 13.
Figure 12. Colonies transformed
Figure 13. PCR Colony EcN transformed with pMUT1-LIRA
The bands in electrophoresis above showed that the DNA size were about 500 bp. However, there was a thin single band appear at around 2000 bp. We suspected that the PCR mix was not specifically amplified the construct. Therefore, we directly subcultured some colonies that had upper single band for plasmid isolation. The isolated plasmid was amplified with LIRA primer. The result shows in Figure 14.
Figure 14. Amplification of isolated pMUT1-LIRA
4. Sequence analysis
According to the electrophoresis result of both PCR Colony and amplification of isolated plasmid, there are no positive desired band (~3000 bp) appear. Therefore, we send the amplified backbone and insert product and some primers for Sanger Sequencing analysis to identify if there was an error in designing the backbone, insert, and their primers.
Plasmid/ InsertSequences
Table 9. pMUT1 sequence result of sequencing
These sequences are then aligned with the designed sequences. The alignment of sequencing result and designed sequence for pMUT1 is shown in Figure 15 and the alignment of sequencing result and designed sequences for LIRAreg2 is shown in Figure 16.
Figure 15. Alignment sequences of pMUT1
Figure 16. Alignment sequences of LIRAreg2
The sequencing result sequences are being observed by BLASTn NCBI. The result of pMUT1 is showing in Figure 17 and LIRAreg2 is showing in Figure 18. From the result of pMUT1, it can be seen that the percent identity is 99,29% belonging to Escherichia coli Nissle 1917. Because of the DNA purity is insufficient during the sequencing process, the sequencing result is not completed, showing in Figure 19. We constructed LIRAreg2 by ourself. Therefore, there is no result appear as “LIRAreg2” in BLASTn searching tool. However, the alignment result of LIRAreg2 sequencing result and designed sequences shows that many mutations happen during the amplification process. This is also giving affect to the unwanted DNA band at around 500 bp size. It can be assumed that the DNA template sequences are affected and mutated by the designed primers. In the other way, we can’t assume completely because the DNA purity is also insufficient during the sequencing process. Therefore, there are many DNA sequences that couldn’t be detected by Sanger sequencing. The quality of DNA purity for sequencing is shown in Figure 20.
Figure 17. pMUT1 BLASTn NCBI result
Figure 18. LIRAreg2 BLASTn NCBI result
Figure 19. Graphic Summary of pMUT1 sequences of sequencing result
Figure 20. Electrophoresis result before sequencing (A) pMUT1 (B) LIRAreg2
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