iGEM 2019 — Team UI_Indonesia

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# Team:UI Indonesia/Notebook

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* [PARTS](https://2019.igem.org/Team%3AUI_Indonesia/Parts)
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* HUMAN PRACTICES

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  [HUMAN
  PRACTICE CATALOGUE](https://2019.igem.org/Team%3AUI_Indonesia/Human_Practice_Catalogue)
* [MEDAL](https://2019.igem.org/Team%3AUI_Indonesia/Medal_Requirements)

# NOTEBOOK

![](https://static.igem.org/mediawiki/2019/1/17/T--UI_Indonesia--arrow.png)

1. [March](#march)
2. [April](#april)
3. [May](#may)
4. [June](#june)
5. [July](#july)
6. [August](#august)
7. [September](#september)
8. [Giant Jamboree](#jamboree)

### 20 March 2019

Team iGEM UI Indonesia was created

### 23 April 2019

Meeting on Biosafety and Biosecurity with Michelle E. Rozo, PhD, from Biosecurity Engagement Programme, U.S. Department of State at the Embassy of the United States of America, Jakarta-Indonesia
![april](https://static.igem.org/mediawiki/2019/7/7e/T--UI_Indonesia--safety-fig-3-meeting.jpg)

### 24 April 2019

iGEM DNA Distribution kit has arrived
![april](https://static.igem.org/mediawiki/2019/a/a9/T--UI_Indonesia--april.png)

### 25 April 2019

Meetings on Project Determination ![april2](https://static.igem.org/mediawiki/2019/1/1e/T--UI_Indonesia--april2.png)

### 11 May 2019

Started designing our primer

### 11 June 2019

Finished the construction of plasmid and designing the primer
![june](https://static.igem.org/mediawiki/2019/7/78/T--UI_Indonesia--june.png)

### 15 June 2019

We started collaboration with iGEM NUS\_Singapore 2019 TEAM

### 16 June 2019

Ordering DNA parts from IDT

### 19 June 2019

Training in the IHVCB Laboratory
![june2](https://static.igem.org/mediawiki/2019/f/fb/T--UI_Indonesia--june2.png)

### 19 July 2019

Ordering DNA parts from IDT

### 20 July 2019

Presentation at Imperial College London
![july](https://static.igem.org/mediawiki/2019/1/18/T--UI_Indonesia--july.png)

### 29 July 2019

Meeting iGEM Ambassador (Mrs. Nurul)
![july2](https://static.igem.org/mediawiki/2019/f/fe/T--UI_Indonesia--july2.png)

### 24 August 2019

Meetup with ITB iGEM TEAM
![August](https://static.igem.org/mediawiki/2019/4/44/T--UI_Indonesia--aug.png)

### 2 September 2019

Finish measuring and making characterization of GFP

### 27 September 2019

Going to iGEM NUS\_Singapore 2019 TEAM for laboratory collaboration
![September](https://static.igem.org/mediawiki/2019/0/0d/T--UI_Indonesia--sep.png)

### **GIANT JAMBOREE**



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# Team:UI Indonesia/Results

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* [HOME](https://2019.igem.org/Team%3AUI_Indonesia)
* TEAM

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  [ATTRIBUTIONS](https://2019.igem.org/Team%3AUI_Indonesia/Attributions)
  [COLLABORATIONS](https://2019.igem.org/Team%3AUI_Indonesia/Collaborations)
* PROJECTS

  [OVERVIEW](https://2019.igem.org/Team%3AUI_Indonesia/Description)
  [DESIGN](https://2019.igem.org/Team%3AUI_Indonesia/Design)
  [EXPERIMENTS](https://2019.igem.org/Team%3AUI_Indonesia/Experiments)
  [NOTEBOOK](https://2019.igem.org/Team%3AUI_Indonesia/Notebook)
  [RESULTS AND
  DISCUSSIONS](https://2019.igem.org/Team%3AUI_Indonesia/Results)
* [PARTS](https://2019.igem.org/Team%3AUI_Indonesia/Parts)
* [MODEL](https://2019.igem.org/Team%3AUI_Indonesia/Model)
* [SAFETY](https://2019.igem.org/Team%3AUI_Indonesia/Safety)
* HUMAN PRACTICES

  [INTEGRATED HUMAN PRACTICES](https://2019.igem.org/Team%3AUI_Indonesia/Human_Practices)
  [EDUCATION AND PUBLIC ENGAGEMENT](https://2019.igem.org/Team%3AUI_Indonesia/Public_Engagement)
  [HUMAN
  PRACTICE CATALOGUE](https://2019.igem.org/Team%3AUI_Indonesia/Human_Practice_Catalogue)
* [MEDAL](https://2019.igem.org/Team%3AUI_Indonesia/Medal_Requirements)

# RESULTS AND DISCUSSION

![](https://static.igem.org/mediawiki/2019/1/17/T--UI_Indonesia--arrow.png)

### **Experiment 1: Characterization of Part OG (Bba\_K769001)**

###### **Part I: Transformation of plasmid containing part OG**

Plasmid pSB1C3+OG was successfully transformed into *E. coli* strain TOP10, DH5a and BL21 (Figure 1). The results were shown under UV light showing bright green color confirming the presence of successful GFP expression.

![](https://static.igem.org/mediawiki/2019/e/e8/T--UI_Indonesia--image031.jpg)

**Figure 1.** The following picture are all bacteria that had been successfully transformed with pSB1C3-OG.

###### **Part II: Expression of GFP**

All bacteria expresses GFP though placed in LB medium with various sodium concentration (Figure 2). After confirming the GFP is successfully expressed by these bacteria, we proceed to SDS PAGE. The result of SDS-PAGE was consistent with our findings where the expression of GFP (26.9 kDA) was indeed increased from low osmolarity to high osmolarity (Figure 3).

![](https://static.igem.org/mediawiki/2019/7/7b/T--UI_Indonesia--image033.png)

**Figure 2.** UV-visualization of pelleted baterial culture expressing GFP.

![](https://static.igem.org/mediawiki/2019/6/6c/T--UI_Indonesia--image035.png)

**Figure 3.** SDS-PAGE result of GFP expression. Red arrows indicate GFP protein (27 kDa). WT indicates wild-type bacteria. Lane 1-5 indicates 85.5 mM (low salt), 171 mM (normal LB broth), 256.5 mM, 342 mM and 427.5 mM sodium concentration treatment respectively

To further characterize these findings, we measured the expression of GFP in MEFL/ particle. In table 1 & 2, the result of our readings are shown. The experiment was performed in duplicate and the average of the value was inserted into the table provided by iGEM that leads to generation of Figure 4. Essentially, all strains of bacteria showed increased fluorescence with increasing NaCl concentrations in LB medium. Interestingly, all bacteria showed to have decreased fluorescence when sodium concentration were increased from 171 mM to 256.6 mM. We had searched multiple papers but questions remain unanswered. There need to be further studies to determine this phenomenon. As expected, when the sodium concentration was to be increased from 256.6 mM to 342 mM, fluorescence readings increased. Fluorescence readings were at its peak when sodium concentration were 427 mM.

**Table 1 and Table 2**. The GFP fluorescence (520nm) of various bacteria in different osmolarity (performed in duplicate)

###### **Table 1.** The GFP fluorescence (520nm) of various bacteria in different osmolarity (performed in duplicate)

| Bacterial Strain | Osmolarity | | | | | | | | | | Blank | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 85.5 mM (duplo) | | 171 mM (duplo) | | 256.5 mM (duplo) | | 342 mM (duplo) | | 427.5 mM (duplo) | |
|  | 1 | 2 | 1 | 2 | 1 | 2 | 1 | 2 | 1 | 2 | 1 | 2 |
| TOP10 | 10597.2 | 10909.4 | 10968.1 | 11472 | 8109.83 | 8554.63 | 7974.81 | 7970.75 | 7363.5 | 7264.01 | 8604.79 | 8634.82 |
| DH5a | 10292.3 | 10296.5 | 10479.7 | 10931.7 | 8506.18 | 9022.21 | 8356.25 | 7920.52 | 7600.73 | 7409.77 |
| BL21 | 9279.19 | 9667.75 | 9781.16 | 9487.33 | 8874.66 | 8835.2 | 8134.98 | 8047.95 | 7437.63 | 7379.81 |

###### **Table 2.** The Absorbance (OD 600nm) of various bacteria in different osmolarity (performed in duplicate)

| Bacterial Strain | Osmolarity |
| --- | --- |
| 85.5 mM (duplo) | | 171 mM (duplo) | | 256.5 mM (duplo) | | 342 mM (duplo) | | 427.5 mM (duplo) | | Blank | |
|  | 1 | 2 | 1 | 2 | 1 | 2 | 1 | 2 | 1 | 2 | 1 | 2 |
| TOP10 | 0.334 | 0.318 | 0.246 | 0.25 | 0.111 | 0.114 | 0.11 | 0.114 | 0.129 | 0.132 | 0.138 | 0.139 |
| DH5a | 0.254 | 0.258 | 0.235 | 0.231 | 0.114 | 0.12 | 0.12 | 0.13 | 0.122 | 0.126 |
| BL21 | 0.329 | 0.326 | 0.434 | 0.421 | 0.417 | 0.422 | 0.124 | 0.123 | 0.117 | 0.121 |

###### **Table 3.** The MELF/particle of various bacteria in different osmolarity.

| Bacterial Strain | Osmolarity | Blank |
| --- | --- | --- |
| 85.5 mM | 171 mM | 256.5 mM | 342 mM | 427.5 mM |
| TOP10 | 12289.01 | 25646.41 | 11945.50 | 26369.48 | 176317.83 | 32432.60 |
| DH5a | 15392.12 | 23838.95 | -7253.12 | 38513.84 | 83015.64 |
| BL21 | 4878.12 | 3791.01 | 903.69 | 38040.71 | 67075.89 |

![](https://static.igem.org/mediawiki/2019/2/2a/T--UI_Indonesia--image041.png)

**Figure 4.** The level of GFP expression in bacteria (TOP10, DH5a, and BL21) containing part BBa\_K769001 after inoculation in LB broth in various sodium concentration for 12 hours.

TOP10 has relatively higher MEFL/particles to be compared with BL21. This is contradicting our thinking that TOP10 will have a lower expression compared to BL21 because BL21 is a protein-expressing-bacteria while TOP10 is bacteria mainly functional for increasing plasmid count (cloning). A possible reason for this phenomenon is that there are differences in ompC promoter regulation in different bacterial strains. (1) OmpR-regulated promoter mediated gene expression even in the absence of its transcription factor. (According to a characterization done by SDU-Denmark (BBa\_R0082), it is found that OmpR-regulated promoter showed a high leaky expression in high copy vectors (e.g. TOP10) compared to low copy vectors (e.g. BL21) confirming our findings in our characterization.(2) From the results, DH5a then BL21 showed a gradual increase in expression with increasing NaCl concentrations in LB medium. This indicates that DH5a and BL21 has a much more efficient control of gene expression with the ompC promoter.

### **Experiment 2: Manufacturing of HB-EGF/EnvZ (HE) + OmpC/GFP (OG)-based diphtheria diagnostic tool system**

###### **Part I: Assembly of part HE into pSB1C3 containing part OG**

The part HB-EGF/EnvZ was successfully combined into plasmid pSB1C3 containing OmpC-GFP. The sequencing result is as follow: [**Sequencing result of pSB1C3 HB+EGF+OmpC-GFP**](http://parts.igem.org/Part%3ABBa_K3088001%3ADesign).

###### **Part II: Expression of HB-EGF/EnvZ (HE) + OmpC/GFP (OG)**

Before we measure the fluorescence of the system, we would like to check the existence of the proteins (HE & OG). We performed SDS-PAGE to measure the reconfirm the size of HE and OG. In Figure 5, we can observe 2 bands that are missed in the wildtype (WT) bacteria. The bands have the size of +/- 90kDA and 43 kDA, respectively. The size of this band is consistent with the size of HE (42.332 kDA) that we already predict by it’s structural modelling. To presence of 90 kDA band also confirms that HE presents in its dimeric form and attached to the membrane. From the thickness of the band, it is shown that TB broth has the thickest band compared to Low Salt LB Medium and Normal LB Medium. In the TB & LB medium, the thickness of the HE band gradually increase from row 1 to row 4, consistent with the duration after IPTG is added. In Figure 5, we can also observe the presence of 43 kDA band. This would confirm that this band is a monomer of our chimeric protein. Based on literature, we know that not all EnvZ will form dimer because of the existence of the dimer-monomer equilibrium.(3) From this we could conclude that each of the 90 kDA band and 43 kDA is our chimeric protein on dimer and monomer structure respectively.

We also perform MagneHis™ on HE to further confirm the presence of HE containing HisTag Figure 5. The resulted MagneHis™ band was really thin confirming HE is indeed expressed in Low Salt Medium and TB medium. Unfortunately, MagneHis™ result on LB medium lane shows several bands which may be due to un-specific binding of the MagneHis™ to HE. Another explanation may be due to saturated concentration of bacteria and thus during the washing part, the washing is not completely clean. Therefore, during the elution, some of the leftover bacterial protein get eluted. It can be observed that HE band is still present.

![](https://static.igem.org/mediawiki/2019/d/d4/T--UI_Indonesia--image043.png)

**Figure 5.** . This shows the SDS-PAGE of TOP10 bacteria grown in TB medium, LB medium and Low Salt LB medium. The lane ‘-’ indicates TOP10 before induced by IPTG. The lane ‘1-4’ indicates sample collected at hour 1 to 4 after IPTG has been added. The lane A, B and C indicates MagneHis™ of TOP10 in TB medium, LB medium, and Low Salt LB medium, respectively. The WT indicates TOP10 wildtype.

Unfortunately in this SDS-PAGE, the GFP protein can not be seen. However, the presence of GFP can be confirmed when the bacteria is pelleted and observed under UV light (Figure 6). The reason the GFP protein can not be observed may be due to short duration of soaking in Page Blue solution.

![](https://static.igem.org/mediawiki/2019/9/9a/T--UI_Indonesia--image045.jpg)

**Figure 6.** The pelleted TOP 10 bacteria transformed with HE-OG plasmid expressing GFP where C1 means Colony 1 and C2 means Colony 2.

###### **Part III: Characterizing HBEGF/EnvZ - OmpC/GFP binding potential with Heparin**

###### **Table 1.** Absorbance 600 Raw Readings.

| Dilution of Heparin Sodium (5000 IU/ml) | TOP10 Bacteria Replicate | | | | | | | | | | | Blank |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 |
| 1/100 dilution (50 IU/ml) | 0.652 | 0.686 | 0.673 | 0.663 | 0.669 | 0.684 | 0.677 | 0.667 | 0.673 | 0.674 | 0.675 | 0.190 |
| 1/1000 dilution (5 IU/ml) | 0.647 | 0.675 | 0.676 | 0.661 | 0.677 | 0.666 | 0.670 | 0.671 | 0.666 | 0.677 | 0.663 | 0.164 |
| 1/10.000 dilution (0.5 IU/ml) | 0.637 | 0.624 | 0.625 | 0.650 | 0.617 | 0.651 | 0.644 | 0.648 | 0.654 | 0.650 | 0.655 | 0.188 |
| Control | 0.650 | 0.642 | 0.635 | 0.640 | 0.638 | 0.646 | 0.634 | 0.647 | 0.669 | 0.668 | 0.667 | 0.169 |

###### **Table 2.** Fluorescence Raw Readings.

| Dilution of Heparin Sodium (5000 IU/ml) | TOP10 Bacteria Replicate | | | | | | | | | | | Blank |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 |
| 1/100 dilution (50 IU/ml) | 10101 | 10160 | 10106 | 10032 | 9991 | 10521 | 10362 | 10399 | 10268 | 10260 | 10190 | 10459 |
| 1/1000 dilution (5 IU/ml) | 10096 | 10235 | 10278 | 10172 | 10611 | 10399 | 10430 | 10373 | 10228 | 10359 | 10190 | 9981 |
| 1/10.000 dilution (0.5 IU/ml) | 10645 | 10241 | 10191 | 10345 | 10444 | 10653 | 10235 | 10076 | 10416 | 10223 | 10208 | 11786 |
| Control | 9628 | 9627 | 9546 | 9824 | 9521 | 9814 | 9647 | 9848 | 9982 | 9910 | 9835 | 9932 |

###### **Table 3.** MEFL/Particle

| Dilution of Heparin Sodium (5000 IU/ml) | TOP10 Bacteria Replicate | | | | | | | | | | | Average | Blank |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 |
| 1/100 dilution (50 IU/ml) | -51.45 | 75.39 | -38.38 | - | -293.63 | 846.90 | 515.65 | 607.56 | 313.25 | - | 143.14 | 235.38 | 29673.81 |
| 1/1000 dilution (5 IU/ml) | -63.57 | 240.83 | 334.49 | 106.30 | 1053.95 | 607.30 | 671.15 | - | - | 509.33 | 147.17 | 400.77 |
| 1/10.000 dilution (0.5 IU/ml) | 1225.55 | 282.29 | 162.43 | 505.39 | 785.92 | - | 256.53 | -111.48 | 662.62 | 227.15 | 189.10 | 418.55 |
| Control | -1133.49 | -1155.13 | -1365.53 | -700.38 | -1414.26 | -716.80 | -1130.38 | -634.61 | -312.21 | -470.30 | -638.80 | -879.26 |

![](https://static.igem.org/mediawiki/2019/a/ad/T--UI_Indonesia--image200.png)

**Figure 7.**GFP expression denoted by MEFL/particle of TOP10 on various concentration of heparin in LB medium.

Our experiment showed the highest MEFL/ particle was observed in TOP10 which was induced with heparin diluted in 1:10000. The MEFL/particle decreases as dilution factor is reduced to 1:1000 and 1:100, respectively. By comparing the MEFL/particle of the heparin-induced-group with the non-heparin-induced-group (control), we can conclude that the induction of heparin significantly increases the number of fluorescence (GFP) expression. This proves that that upon binding of heparin sodium to our chimeric protein (HB-EGF/EnvZ), more OmpR is phosphorylated that leads to increased activation of OmpC promoter and the GFP gene downstream the OmpC promoter is successfully expressed. Our chimeric protein works !

Fluorescence reading of our blank shows a significant amount of fluorescence which might be due to the background fluorescence of the medium emits similar wavelength with GFP.4

###### **Statistical Analysis**

We then try to perform statistical analysis using SPSS Statistic 24. We performed One Way ANOVA (since our data distribution is normal and more than 2 groups). We then proceed with Post Hoc Bonferoni because our data is homogenous. From the result, there is statistical significance (p<0.05) between sample without heparin treatment and sample treated with heparin (Figure 4). Data between treatment (1:100, 1:1000, and 1:10000) does not show any significance may be due to the receptor is oversaturated with the ligands.

![](https://static.igem.org/mediawiki/2019/thumb/f/ff/T--UI_Indonesia--statanalysis.png/799px-T--UI_Indonesia--statanalysis.png)

**Figure 4.** The statistical calculation of our result using One Way Anova comparing the sample without heparin treatment to samples treated with heparin.

###### **Discussion**

Initially, our hypothesis was the more heparin is present, the expression of GFP will increase in a similar manner. However, the expression of GFP is increased when the dilution factor is increased from 1/100, 1/1000, and 1/10000. The increase is quite significant between 1/1000 and 1/10000. However, we suspect that decrease of GFP expression as the dilution factor is decrease may be caused by antimicrobial activity of heparin.5,6 According to Rosett W3, the heparin concentration needed to inhibit bacterial growth of Escherichia coli is ≤ 500 U/ml. Our 1/100 concentration of heparin sodium 5000 U/ml is 50 U/ml. The antimicrobial activity of heparin might inhibit the growth and expression of GFP and the concentration of heparin needed to induce the maximum expression of GFP is ≤ 0.5 U/ml (1/10.000 dilution).

### **Experiment 3: Improvement and characterization of part DT18 (Bba\_K2607000)**

###### **Part I: Assembly of part DT18 and DT19 into pQE80L**

The transformation of DT18 was very successfully whereas unfortunately the transformation of DT19 failed. 19 colonies of DT18 transformation result were amplified using PCR and the result is shown in Figure 1. For DT19. the sequence.

![](https://static.igem.org/mediawiki/2019/9/96/T--UI_Indonesia--image047.png)

###### **Part II: Expression of DT18 and DT19**

DT19 was not successfully transformed into bacteria so we could not characterize it. However. all the result of DT18 expression were showing negative results.

###### **References**

1. Levskaya A, Chevalier AA, Tabor JJ, Simpson ZB, Lavery LA, Levy M, et al. Engineering Escherichia coli to see light. Nature [Internet]. 2005 Nov 23 [cited 2019 Oct 16];438(7067):441–2. Available from:http://www.nature.com/articles/nature04405
2. SDU Denmark. BBa\_R0082. iGEM [Internet].http://parts.igem.org/Part:BBa\_R0082#Contribution:\_Hamburg\_2016
3. Khorchid A, Inouye M, Ikura M. Structural characterization of Escherichia coli sensor histidine kinase EnvZ: the periplasmic C-terminal core domain is critical for homodimerization. Biochem J [Internet]. 2005 Jan 1 [cited 2019 Oct 20];385(Pt 1):255–64. Available from:http://www.ncbi.nlm.nih.gov/pubmed/15357641
4. Electronic Y. Spreading generally increases with lower cell ­ cell adhesion and higher cell ­ substrate adhesion . However , strong substrate adhesion can adversely affect cell movement , and modeling suggests an optimum substrate adhesiveness for spreading ( Xu et al. Phys Biol. 2016;12(2010):11–2.
5. Weijmer MC. Superior antimicrobial activity of trisodium citrate over heparin for catheter locking. Nephrol Dial Transplant. 2002 Dec 1;17(12):2189–95.
6. Rosett W, Hodges GR. Antimicrobial activity of heparin. J Clin Microbiol [Internet]. 1980 Jan [cited 2019 Oct 21];11(1):30–4. Available from: <http://www.ncbi.nlm.nih.gov/pubmed/6766462>