Difference between revisions of "Part:BBa K3490001"

Latest revision as of 06:52, 17 October 2021

IPTG inducible NOS, over-express csgD and csgA

This year, our team aims to reduce intraocular pressure (IOP) through the contact lens with engineered E. coli that can produce Nitric Oxide (NO). We aimed to make our plasmid express NOS and support our bacteria to bind to the contact lenses. Since E.coli doesn't produce nitric oxide, we ordered a sequence containing a lacO-T7 promoter, B0034 RBS, and bsNOS. Then, we ligate the sequence by inserting it into the PUC plasmid and combining it with another sequence (pLac, RBS, csgA, RBS, csgD, LacI) from IDT and transformed into E. coli DH5-Alpha. By doing so, NOS can convert L-arginine into nitric oxide, thus releasing nitric oxide in the eyes.^[1]

To ensure the plasmid contains those two sequences, we conduct PCR with each sequence’s primer separately. Fig.1 shows that the plasmid contains both of the desired sequences.

Fig. 1. Confirmation of our construction by PCR. M: Marker; Lane 1: nos (~2400 bp); Lane 2: csgA-csgD (~1500 bp).

In order to test the function of the T7 promoter, we transformed the plasmid into BL21(DE3). Next, to observe the effects of various IPTG concentrations on NOS expression, we performed SDS-PAGE with different IPTG concentrations. The bacteria is cultured for two hours and induced with IPTG for 12 hours. In Fig. 2 we can observe that the first to the third lane express a similar thin band. Meanwhile, we can see a thicker band in the fourth lane, which means it has a higher protein expression when the IPTG concentration is 1 mM.

Fig. 2. SDS-PAGE of E.coli BL21(DE3) with different concentrations of IPTG. M: Marker; Lane 1: 0.1 mM IPTG; Lane 2: 0.05 mM IPTG; Lane 3: 0.025 mM IPTG; Lane 4: 1 mM IPTG. The arrow from top to bottom indicates NOS (~40kDa), CsgD (~24kDa), and CsgA (~17kDa).

As we decided to use an IPTG inducible system, we conducted an experiment to determine whether IPTG concentration and induction time can control the production of nitric oxide. Therefore, we test the IPTG system by using different concentrations and induced at different times. Here, we are using E. coli BL21(DE3) strain. As seen in Fig. 3, the nitric oxide production is both time dependent and IPTG dependent. Thereby, we can observe whether the IPTG inducible system can effectively produce nitric oxide in a certain induction time.

Fig. 3. NOS induced by IPTG with different concentrations at different induced times.

Improvement: Overview

One of the goals we want to achieve is to attach our bacteria to the inner chamber of the contact lens. Here, we used csgD, a master regulator of biofilm production; and csgA, a major subunit of the curli fimbriae. Based on research, curli fibers are involved in adhesion to surfaces, cell aggregation, and biofilm formation.^[2] As a transcription factor of curli proteins, CsgD can regulate the expression of csgA, leading to biofilm production.
Therefore, we are trying to improve BBa_K805015 from the 2017 iGEM TAS-Taipei Team^[3] to obtain maximum results of biofilm production and improve the biofilm binding affinity. So, the bacteria will stay attached to the contact lenses despite any damages.
We hypothesized that by adding csgA and pLac to the biobrick design, the amount of biofilm produced could be increased. Thus, the overproduction of biofilm can initiate the bacteria to bind securely to the contact lenses. Therefore, we add csgA to the biobrick design and change the promoter into pLac as an improvement.

Fig.4. A schematic of our biobrick construction.

Experiment Results

First, we ran SDS-PAGE to identify and quantify the protein expression of CsgA and CsgD. We cultured the bacteria for 2 hours, then added IPTG to induce for 12 hours long, and adjusted the OD₆₀₀ value to three. As a comparison, we used plasmid that contains pLac-csgD on BW25113, improved parts that include NOS, csgA, and csgD on BW25113, and using PCA24N as control. After that, we transformed into E. coli BL21(DE3) strain. The expected protein size of CsgD is around 24 kDa and CsgA around 17 kDa. The results below have shown the outcome we expected for CsgA and CsgD protein expression.

Fig.5. SDS-PAGE of E.coli BL21(DE3). M: Marker; Lane 1: Wild type csgD in PCA24N; Lane 2: Wild type BW25113 (control); Lane 3: Knockout csgD in PCA24N; Lane 4: Knockout BW25113 (control); Lane 5: BL21(DE3)-nos-csgA-csgD. The arrow from top to bottom indicates NOS (~40kDa), CsgD (~24kDa), and CsgA (~17kDa).

Next, to prove that our amount of biofilm production is increasing, we did a test using congo red^[4] dye to observe the curli expression. We compared the absorbance value of BBa_K805015 and BBa_K3490001 to see whether the amount of biofilm production increases or not. If the biofilm amount increases, the color of the solution will appear to be darker. So, after overnight culture, we add congo red dye to all the samples. Then we centrifuge to separate the supernatant and precipitate (pellet). By using a microplate reader, we can measure the absorbance value at 500 nm (congo red) and normalized by 600 nm wavelength which represents the amount of bacteria.

Fig.6. Amount of biofilm being produced by bacteria with different genetic backgrounds at different times.

However, only testing the amount of biofilm production and congo red staining is not enough to fully support our aims that enhance biofilm production and improve the binding affinity of the bacteria. Therefore, we conduct another experiment to compare the binding affinity of bacteria among control, csgD, and csgA-csgD. We presume that binding affinity is determined by the ability of bacteria to remain attached to surfaces regardless of the external forces applied. Here, we threw a book from different heights and measuring its OD₆₀₀ value. By doing so, we can determine the concentration that represents the binding affinity.

Fig.7. (A) The formula and graph of the experiment. (The weight of book is 1.6 kilogram, the collision is considered as completely inelastic collision, and the collision time is presumed as 0.1 second.) (B) The binding affinity of control, csgD, and csgA-csgD with given force from different heights.

As seen in the graph above, csgA-csgD shows an increase in OD₆₀₀ value when a greater force is given. Hence, we are able to prove that not only enhances the production of biofilm, but our engineered bacteria can also improve its binding affinity. Therefore, we can conclude that we have successfully improved the previous biobrick BBa_K805015 by adding csgA and changing the promoter into BBa_K3490001.

Reference

[1] BRENDA - Information on EC 1.14.13.39 - nitric-oxide synthase (NADPH). Brenda-enzymes.org. https://www.brenda-enzymes.org/enzyme.php?ecno=1.14.13.39#pH%20OPTIMUM. Published 2020. Accessed September 9, 2020.
[2] Barnhart MM, Chapman MR. Curli Biogenesis and Function. Annual Review of Microbiology. 2006;60(1):131-147.
[3] Part:BBa K805015 - parts.igem.org. Igem.org. https://parts.igem.org/Part:BBa_K805015. Published 2013. Accessed September 21, 2020.
[4] Jones CJ, Wozniak DJ. Congo Red Stain Identifies Matrix Overproduction and Is an Indirect Measurement for c-di-GMP in Many Species of Bacteria. c-di-GMP Signaling. 2017:147-156.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 3923
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 1826
Illegal SapI.rc site found at 2144

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K3490001 short</partinfo>
-This year, our team aims to reduce intraocular pressure (IOP) through the contact lens with engineered E. coli that can produce Nitric Oxide (NO). We aimed to make our plasmid express NOS and support our bacteria to bind to the contact lenses. Since E.coli doesn't produce nitric oxide, we ordered a sequence containing a lacO-T7 promoter, B0034 RBS, and bsNOS. Then, we ligate the sequence by inserting it into the PUC plasmid and combining it with another sequence (pLac, RBS, csgA, RBS, csgD, LacI) from IDT and transformed into E. coli DH5-Alpha. By doing so, NOS can convert L-arginine into nitric oxide, thus releasing nitric oxide in the eyes.
+This year, our team aims to reduce intraocular pressure (IOP) through the contact lens with engineered <i>E. coli</i> that can produce Nitric Oxide (NO). We aimed to make our plasmid express NOS and support our bacteria to bind to the contact lenses. Since <i>E.coli</i> doesn't produce nitric oxide, we ordered a sequence containing a lacO-T7 promoter, B0034 RBS, and bsNOS. Then, we ligate the sequence by inserting it into the PUC plasmid and combining it with another sequence (<i>pLac</i>, RBS, <i>csgA</i>, RBS, <i>csgD</i>, <i>LacI</i>) from IDT and transformed into <i>E. coli</i> DH5-Alpha. By doing so, NOS can convert L-arginine into nitric oxide, thus releasing nitric oxide in the eyes.<sup>[1]</sup>
 To ensure the plasmid contains those two sequences, we conduct PCR with each sequence’s primer separately. Fig.1 shows that the plasmid contains both of the desired sequences.
@@ Line 9: / Line 9: @@
 <br>
 <div style="width=100%; display:flex; align-items: center; justify-content: center;">
-<img src="https://static.igem.org/mediawiki/parts/d/de/T--NCKU_Tainan--BBa_K3490001.png" style="width:35%;">
+<img src="https://static.igem.org/mediawiki/parts/d/de/T--NCKU_Tainan--BBa_K3490001.png" style="width:50%;">
 </div>
-<br>Fig. 1. Confirmation of our construction by PCR. M: Marker; Lane 1: NOS (~2400 bp); Lane 2: CsgA-CsgD (~1500 bp).
+<p align="center">Fig. 1. Confirmation of our construction by PCR. M: Marker; Lane 1: <i>nos</i> (~2400 bp); Lane 2: <i>csgA</i>-<i>csgD</i> (~1500 bp).</p>
 </html>
-In order to test the function of the T7 promoter, we transformed the plasmid into BL21(DE3). Next, to observe the effects of various IPTG concentrations on NOS expression, we performed SDS-PAGE with different IPTG concentrations. The bacteria is cultured for two hours and induced with IPTG for 12 hours. In Fig. 2 we can observe that the first to the third lane express a similar thin band. Meanwhile, we can see a thicker band in the fourth lane, which means it has a higher protein expression when the IPTG concentration is 1 mM.
+<br>
+<br>In order to test the function of the T7 promoter, we transformed the plasmid into BL21(DE3). Next, to observe the effects of various IPTG concentrations on NOS expression, we performed SDS-PAGE with different IPTG concentrations. The bacteria is cultured for two hours and induced with IPTG for 12 hours. In Fig. 2 we can observe that the first to the third lane express a similar thin band. Meanwhile, we can see a thicker band in the fourth lane, which means it has a higher protein expression when the IPTG concentration is 1 mM.
 <html>
@@ Line 20: / Line 21: @@
 <img src="https://static.igem.org/mediawiki/parts/6/6f/T--NCKU_Tainan--BBa_K3490001-nos-sds_phage.png" style="width:35%;">
 </div>
-<br>Fig. 2. SDS-PAGE of E.coli BL21(DE3) with different concentrations of IPTG. M: Marker; Lane 1: 0.1 mM IPTG; Lane 2: 0.05 mM IPTG; Lane 3: 0.025 mM IPTG; Lane 4: 1 mM IPTG. The arrow from top to bottom indicates NOS (~40kDa), CsgD (~24kDa), and CsgA (~17kDa).
+<p align="center">Fig. 2. SDS-PAGE of <i>E.coli</i> BL21(DE3) with different concentrations of IPTG. M: Marker; Lane 1: 0.1 mM IPTG; Lane 2: 0.05 mM IPTG; Lane 3: 0.025 mM IPTG; Lane 4: 1 mM IPTG. The arrow from top to bottom indicates NOS (~40kDa), CsgD (~24kDa), and CsgA (~17kDa). </p>
 </html>
-As we decided to use an IPTG inducible system, we conducted an experiment to determine whether IPTG concentration and induction time can control the production of nitric oxide. Therefore, we test the IPTG system by using different concentrations and induced at different times. Here, we are using E. coli BL21(DE3) strain. As seen in Fig. 3, the nitric oxide production is both time dependent and IPTG dependent. Thereby, we can observe whether the IPTG inducible system can effectively produce nitric oxide in a certain induction time.
+<br>As we decided to use an IPTG inducible system, we conducted an experiment to determine whether IPTG concentration and induction time can control the production of nitric oxide. Therefore, we test the IPTG system by using different concentrations and induced at different times. Here, we are using <i>E. coli</i> BL21(DE3) strain. As seen in Fig. 3, the nitric oxide production is both time dependent and IPTG dependent. Thereby, we can observe whether the IPTG inducible system can effectively produce nitric oxide in a certain induction time.
 <html>
    <br>
    <div style="width=100%; display:flex; align-items: center; justify-content: center;">
-     <img src="https://static.igem.org/mediawiki/parts/7/71/T--NCKU_Tainan--BBa_K3490001-IPTG.png" style="width:35%;">
+     <img src="https://2020.igem.org/wiki/images/d/d3/T--NCKU_Tainan--results-iptg2.png" style="width:50%;">
      </div>
-   <br>Fig. 3. NOS induced by IPTG with different concentrations at different induced times.
+   <p align="center">Fig. 3. NOS induced by IPTG with different concentrations at different induced times.</p>
 </html>
-After confirming the production of nitric oxide can be induced by IPTG, we test the kinetic of NOS. With Fig.4, we cultured the bacteria for 12 hours and are induced with 0.1 mM IPTG for 2 hours. We then applied the homogenized bacteria and substrate to the cornea of the Porcine eye to observe whether the concentration of nitric oxide will increase in the eye or not. The substrate runs out within 20 min and produces approximately 1.6 nmol nitric oxide. Through the graph below, we can observe that when there is 1.6 nmol nitric oxide in the porcine eye, it will diffuse into the cornea and the concentration of nitric oxide will increase six times higher when it reaches aqueous humour.
+<br>
+<br><b style="font-size:1.5rem">Improvement: Overview</b>
+<br>
+<br>One of the goals we want to achieve is to attach our bacteria to the inner chamber of the contact lens. Here, we used <i>csgD</i>, a master regulator of biofilm production; and <i>csgA</i>, a major subunit of the curli fimbriae. Based on research, curli fibers are involved in adhesion to surfaces, cell aggregation, and biofilm formation.<sup>[2]</sup> As a transcription factor of curli proteins, CsgD can regulate the expression of <i>csgA</i>, leading to biofilm production.
+<br>Therefore, we are trying to improve BBa_K805015 from the 2017 iGEM TAS-Taipei Team<sup>[3]</sup> to obtain maximum results of biofilm production and improve the biofilm binding affinity. So, the bacteria will stay attached to the contact lenses despite any damages.
+<br> We hypothesized that by adding <i>csgA</i> and <i>pLac</i> to the biobrick design, the amount of biofilm produced could be increased. Thus, the overproduction of biofilm can initiate the bacteria to bind securely to the contact lenses. Therefore, we add <i>csgA</i> to the biobrick design and change the promoter into <i>pLac</i> as an improvement.
 <html>
    <br>
    <div style="width=100%; display:flex; align-items: center; justify-content: center;">
-     <img src="https://static.igem.org/mediawiki/parts/2/2f/T--NCKU_Tainan--BBa_K3490001_figure_4.png" style="width:35%;">
+     <img src="https://2020.igem.org/wiki/images/1/1b/T--NCKU_Tainan--Improvment_CsgDA.gif" style="width:50%;">
      </div>
-   <br>Fig. 4. NOS activity at different working times to observe when the substrate will be depleted.
+   <p align="center">Fig.4. A schematic of our biobrick construction. </p>
 </html>
+<br><b style="font-size:1.5rem">Experiment Results</b>
+<br>
+<br>First, we ran SDS-PAGE to identify and quantify the protein expression of CsgA and CsgD. We cultured the bacteria for 2 hours, then added IPTG to induce for 12 hours long, and adjusted the OD<sub>600</sub> value to three. As a comparison, we used plasmid that contains <i>pLac-csgD</i> on BW25113, improved parts that include <i>NOS</i>, <i>csgA</i>, and <i>csgD</i> on BW25113, and using PCA24N as control. After that, we transformed into <i>E. coli</i> BL21(DE3) strain. The expected protein size of CsgD is around 24 kDa and CsgA around 17 kDa. The results below have shown the outcome we expected for CsgA and CsgD protein expression.
 <html>
    <br>
    <div style="width=100%; display:flex; align-items: center; justify-content: center;">
-     <img src="https://static.igem.org/mediawiki/parts/1/11/T--NCKU_Tainan--BBa_K3490001-kinetic.png" style="width:35%;">
+     <img src="https://static.igem.org/mediawiki/parts/1/15/T--NCKU_Tainan--BBa_K3490001-csgd.png" style="width:35%;">
      </div>
-   <br>Fig. 5. The concentration of NO is six times higher after the bacteria and substrate is diffused into the porcin eye.
+   <p align="center">Fig.5. SDS-PAGE of <i>E.coli</i> BL21(DE3). M: Marker; Lane 1: Wild type <i>csgD</i> in PCA24N; Lane 2: Wild type BW25113 (control); Lane 3: Knockout <i>csgD</i> in PCA24N; Lane 4: Knockout BW25113 (control); Lane 5: BL21(DE3)-<i>nos</i>-<i>csgA</i>-<i>csgD</i>. The arrow from top to bottom indicates NOS (~40kDa), CsgD (~24kDa), and CsgA (~17kDa). </p>
 </html>
+<br>
-We are taking advantage of the IPTG inducible system to control NOS expression, thus producing nitric oxide. From all the experimental results above, we have proved that our system is functioning well. Starting from NOS construction, confirm NOS expression, until NOS functional test, we have provided convincing results to support our ideas.
+<br> Next, to prove that our amount of biofilm production is increasing, we did a test using congo red<sup>[4]</sup> dye to observe the curli expression. We compared the absorbance value of BBa_K805015 and BBa_K3490001 to see whether the amount of biofilm production increases or not. If the biofilm amount increases, the color of the solution will appear to be darker. So, after overnight culture, we add congo red dye to all the samples. Then we centrifuge to separate the supernatant and precipitate (pellet). By using a microplate reader, we can measure the absorbance value at 500 nm (congo red) and normalized by 600 nm wavelength which represents the amount of bacteria.
+<html>
+  <br>
+  <div style="width=100%; display:flex; align-items: center; justify-content: center;">
+    <img src="https://static.igem.org/mediawiki/parts/d/d6/T--NCKU_Tainan--congo_red_ver2.png" style="width:40%;">
+    </div>
+  <p align="center">Fig.6. Amount of biofilm being produced by bacteria with different genetic backgrounds at different times.  </p>
+</html>
+<br>
+<br>However, only testing the amount of biofilm production and congo red staining is not enough to fully support our aims that enhance biofilm production and improve the binding affinity of the bacteria. Therefore, we conduct another experiment to compare the binding affinity of bacteria among control, <i>csgD</i>, and <i>csgA</i>-<i>csgD</i>. We presume that binding affinity is determined by the ability of bacteria to remain attached to surfaces regardless of the external forces applied. Here, we threw a book from different heights and measuring its OD<sub>600</sub> value. By doing so, we can determine the concentration that represents the binding affinity.
+<html>
+  <br>
+  <div style="width=100%; display:flex; align-items: center; justify-content: center;">
+    <img src="https://2020.igem.org/wiki/images/d/d3/T--NCKU_Tainan--measure-pic8.png" style="width:60%;">
+    </div>
+  <p align="center">Fig.7. (A) The formula and graph of the experiment. (The weight of book is 1.6 kilogram, the collision is considered as completely inelastic collision, and the collision time is presumed as 0.1 second.) (B) The binding affinity of control, <i>csgD</i>, and <i>csgA-csgD</i> with given force from different heights.</p>
+</html>
+<br>
+<br>As seen in the graph above, <i>csgA</i>-<i>csgD</i> shows an increase in OD<sub>600</sub> value when a greater force is given. Hence, we are able to prove that not only enhances the production of biofilm, but our engineered bacteria can also improve its binding affinity. Therefore, we can conclude that we have successfully improved the previous biobrick BBa_K805015 by adding <i>csgA</i> and changing the promoter into BBa_K3490001.
+<br>
+<br><b style="font-size:1.5rem">Reference</b>
+<br>
+<br>[1] BRENDA - Information on EC 1.14.13.39 - nitric-oxide synthase (NADPH). Brenda-enzymes.org. https://www.brenda-enzymes.org/enzyme.php?ecno=1.14.13.39#pH%20OPTIMUM. Published 2020. Accessed September 9, 2020.
+<br>[2] Barnhart MM, Chapman MR. Curli Biogenesis and Function. Annual Review of Microbiology. 2006;60(1):131-147.
+<br>[3] Part:BBa K805015 - parts.igem.org. Igem.org. https://parts.igem.org/Part:BBa_K805015. Published 2013. Accessed September 21, 2020.
+<br>[4] Jones CJ, Wozniak DJ. Congo Red Stain Identifies Matrix Overproduction and Is an Indirect Measurement for c-di-GMP in Many Species of Bacteria. c-di-GMP Signaling. 2017:147-156.