iGEM 2019 — Team Bilkent-UNAMBG

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# Team:Bilkent-UNAMBG/Notebook

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![](https://static.igem.org/mediawiki/2019/2/25/T--Bilkent-UNAMBG--labnotebookheader.png)

### February

* We held our first meeting with the new members.

### March

* Brainstorming for the project.
* We realized that everybody in the team is interested in biomedical applications of synthetic biology.
* We arranged workshops for the new members.

### April

* Safety training for the team.
* Experimental training for the new members.
* We started to seek sponsorships.
* We started literature search based on metabolic disorders and therapeutics.

### May

* A survey on metabolic disorders were prepared and published.
* We started to do comprehensive literature research for Diabetes Mellitus.
* Potential insulin analogs such as viral insulin like peptides were evaluated.
* We held a meeting with Prof. Dr. Okan Bülent Yıldız, from the Department of Internal Medicine Endocrinology and Metabolism of Hacettepe University.
* We decided to make insulin analog secreting probiotic bacteria as our project subject based on the ideas we get from Prof. Dr. Okan Bülent Yıldız and public survey results.

### June

* Our team joined to diabetes patient education sessions at Hacettepe University.
* The team met in the lab for the first time.
* We started to clone sfGFP and TEV protease.
* Started to mathematical modelling.
* Our team wrote a story book on synthetic biology for kids. We sent an English version of our story to the teams contacted us. The teams that translated our story into their languages.
* We designed a quiet book for preschool kids to demonstrate basic principles of synthetic biology.
* We started to our journal clubs.

### July

* We initiated a collaboration. We sent an English version of our story book to other teams. The teams translated our story into their languages.
* Our IDT constructs arrived and we finished the cloning of them.

### August

* We made part characterization.
* The cloning of all constructs were completed.
* Double transformation were done to B strain.
* We started to induction and kinetic measurement experiments.
* We checked for the surface displays of our constructs.

### September

* We got our sponsorships from Traveyo company and Dyslexia and Learning Disabilities Foundation.
* We arranged a Skype meeting with ULaVerne team.
* Protein purification for our working constructs.

### October

* We visited 4th grade students to introduce the concept of synthetic biology. We demonstrated them the basics of synthetic biology with our quiet book and the Washington iGEM Teams’ activity booklet.
* Supported our modelling with the experimental data.
* We collaborated with IIT Madras iGEM team for their ‘Language Project 2.0’.
* We filmed a short parody music clip.
* We visited Başkent Deaf Sports Foundation and explain them to our project with the grammar of Turkish Sign Language.
* Finished the wiki.

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# Team:Bilkent-UNAMBG/Experiments

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[Human Practices](https://2019.igem.org/Team%3ABilkent-UNAMBG/Human_Practices)[Education & Engagement](https://2019.igem.org/Team%3ABilkent-UNAMBG/Public_Engagement)[Safety](https://2019.igem.org/Team%3ABilkent-UNAMBG/Safety)

![](https://static.igem.org/mediawiki/2019/7/76/T--Bilkent-UNAMBG--experiments.jpg)

### Protocols

* [Bilkent-UNAMBG 2019 Protocols](https://static.igem.org/mediawiki/2019/d/d3/T--Bilkent-UNAMBG--protocols.pdf)

### Experiments

* [Construction of SCIA Display Casettes](https://static.igem.org/mediawiki/2019/2/27/T--Bilkent-UNAMBG--experiment1.pdf)
* [Construction of inducible TEV protease Display plasmids](https://static.igem.org/mediawiki/2019/d/de/T--Bilkent-UNAMBG--experiment2.pdf)
* [Construction of Constitutive sfGFP Display plasmids](https://static.igem.org/mediawiki/2019/c/c6/T--Bilkent-UNAMBG--experiment3.pdf)
* [Induction & Kinetic Measurements](https://static.igem.org/mediawiki/2019/3/31/T--Bilkent-UNAMBG--experiment4.pdf)

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Copyright © Bilkent-UNAMBG. Designed by Baturalp Uyar.

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# Team:Bilkent-UNAMBG/Results

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[Human Practices](https://2019.igem.org/Team%3ABilkent-UNAMBG/Human_Practices)[Education & Engagement](https://2019.igem.org/Team%3ABilkent-UNAMBG/Public_Engagement)[Safety](https://2019.igem.org/Team%3ABilkent-UNAMBG/Safety)

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# **Goals**

**\***Finishing all of our clonings.

**\***Testing our release mechanism.

**\***Testing insulin synthesis.

**\***Testing our insulin activity.

### **Clonings**

Cloning was a big part of our project because we had a significant amount of constructs.

### **TEV Cloning**

We cloned the TEV protease fused with the Ag43 autotransporter into 3 different plasmids, high, mid and low copy. These plasmids have origins, pBR322, p15A and pSC101 respectively.

![](https://static.igem.org/mediawiki/2019/3/3d/T--Bilkent-UNAMBG--TEVHighResult.png)

**Figure 1: The plasmid design and PCR images of insert and backbone of TEV high copy.**

![](https://static.igem.org/mediawiki/2019/5/50/T--Bilkent-UNAMBG--LowCo.png)

**Figure 2: The plasmid design and PCR images of insert and backbones of TEV low copy.**

![](https://static.igem.org/mediawiki/2019/2/24/T--Bilkent-UNAMBG--MidCopy.png)

**Figure 3: The plasmid design and PCR images of insert and backbone of TEV mid copy.**

### **sfGFP Cloning**

We aimed to clone sfGFP along with the Ag43 autotransporter into 3 different plasmids high, mid and low copy. These also have pBR322, p15A and pSC101 origins respectively. Unfortunately we were not able to clone the sfGFP high copy construct after many attempts we decided to scratch it.

![](https://static.igem.org/mediawiki/2019/1/19/T--Bilkent-UNAMBG--LowsfGFP.png)

**Figure 4: The plasmid design, PCR images of insert and backbone of sfGFP mid copy and RFU/OD600 graph of sfGFP mid copy.**

![](https://static.igem.org/mediawiki/2019/e/e5/T--Bilkent-UNAMBG--MidCopysfGFP.png)

**Figure 5: The plasmid design, PCR images of insert and backbone of sfGFP low copy and RFU/OD600 graph of sfGFP low copy.**

### **SCI Cloning**

We synthesized our SCI sequences from IDT we cloned them into pET22b plasmids which have the high copy origin pBR322.
![](https://static.igem.org/mediawiki/2019/9/92/T--Bilkent-UNAMBG--SCI57Patent.jpg)

**Figure 6: The plasmid design and colony PCR verifications of the SCI construct SCI57 with a linker peptide.**

![](https://static.igem.org/mediawiki/2019/f/f9/T--Bilkent-UNAMBG--SCIRajpal15.jpg)

**Figure 7: The plasmid design and colony PCR verifications of the SCI construct SCI57 with a linker peptide (Rajpal 15)**

![](https://static.igem.org/mediawiki/2019/6/60/T--Bilkent-UNAMBG--SCIRajpal27.jpg)

**Figure 8: The plasmid design and colony PCR verifications of the SCI construct SCI57 with a linker peptide (Rajpal 27)**

![](https://static.igem.org/mediawiki/2019/5/54/T--Bilkent-UNAMBG--sc%C4%B157sc%C4%B157.jpg)

**Figure 9: The plasmid design and colony PCR verifications of the SCI construct SCI57 with a linker peptide (SCI 57)**

![](https://static.igem.org/mediawiki/2019/f/fa/T--Bilkent-UNAMBG--wildtyperajpal15.jpg)

**Figure 10: The plasmid design and colony PCR verifications of the SCI construct, wild type with a linker peptide.**

![](https://static.igem.org/mediawiki/2019/a/a4/T--Bilkent-UNAMBG--WildTypeLinkerPeptide.jpg)

**Figure 11: The plasmid design and colony PCR verifications of the SCI construct wild type with a linker peptide (Rajpal 15).**

![](https://static.igem.org/mediawiki/2019/0/0d/T--Bilkent-UNAMBG--wildtyperajpal27.jpg)

**Figure 12: The plasmid design and colony PCR verifications of the SCI construct wild type with a linker peptide (Rajpal 27).**

![](https://static.igem.org/mediawiki/2019/3/38/T--Bilkent-UNAMBG--wildtypesci57.jpg)

**Figure 13: The plasmid design and colony PCR verifications of the SCI construct wild type with a linker peptide (SCI 57).**

In order to see the expression and display level of our sfGFP Ag43 constructs, we performed Heat Release experiment according to our Heat Release Protocol. Our constructs were transformed into B strain BL21 Marionette cells prior to the experiment. We performed the experiment on these cells since we were also planning to perform arabinose induction on BL21 Marionette cells. The cells were grown in LB for 16 hours.

The cells display sfGFP on the cell surface via the Ag43 autotransporter. When these cells are heated briefly, they release the displayed sfGFP along with the truncated passenger domain of Ag43 into the supernatant (1,2). The supernatant fluorescence was measured with the M5 spectrophotometer.

As seen from the graph given in Figure 1, there is a 39 fold difference between heat treated pZs sfGFP Ag43 and heat untreated pZs sfGFP Ag43. There is a 21 fold difference between heat treated pZa sfGFP Ag43 and untreated pZa sfGFP Ag43.

![](https://static.igem.org/mediawiki/2019/3/3f/T--Bilkent-UNAMBG--heatreleaselb.png)

**Figure 14: Fluorescence Data of sfGFP Ag43 Constructs (Heat Release)**

RFU stands for relative fluorescence unit and a.u. stands for arbitrary unit. Heat (+) means heat-treated. Heat (-) means heat-untreated. pZs sfGFP Ag43 signifies the cells having the plasmid pZs sfGFP Ag43. pZa sfGFP Ag43 signifies the cells having the plasmid pZa sfGFP Ag43.

### **Characterization of sfGFP Ag43 expression via Growth & Fluorescence Measurement**

In order to characterize sfGFP Ag43 expression while cells are growing, we performed growth & fluorescence measurement experiment according to our growth and fluorescence measurement protocol. The measurement was done on two different cell strains to which our construct were transformed previously. Our purpose of characterizing each construct in two different cell strain was to obtain a comparative result for sfGFP Ag43 expression to see how strain type affects the expression.

Cells were grown in LB for 16 hours and diluted to 1:100 ratio. Starting from the dilution time, we measured the expression and display of sfGFP every 30 minutes for 6 hours.

In Figure 15 and Figure 16, RFU/OD600 (a.u.) data for our constructs pZs sfGFP Ag43 and pZa sfGFP Ag43 are shown. In Figure 17, RFU/OD600 (a.u.) data for empty DH5α (MG1655) and B strain (BL21 DE3) cells are shown. We mean wild type cells without plasmid transformation by “empty cells”.

As it can be seen from the graphs in Figure 15, Figure 16 and Figure 17 the RFU/OD600 (a.u.) value is almost constant starting from the minute 120. Because the OD600 is constantly increasing we can extrapolate that the fluorescence also increases, as we expected. The unconstant pattern observed in the first 120 minutes might be due to experimental errors. On the other hand, it might be related to cell metabolism. The negative values in Figure 17, might be due to experimental errors. An accurate measurement may not have been possible since cell amount was low in the beginning, not higher than 0.2 OD600.

Figure 18, Figure 19 and Figure 20 are the growth curves for the cells. DH5α (MG1655) has a higher value than B strain (BL21 DE3) in all graphs.

Comparing Figure 19 to Figure 20, cells with pZa sfGFP Ag43 plasmid reached a higher OD600 value. The reason might be the same with the cells having pZs sfGFP Ag43 plasmid.

![](https://static.igem.org/mediawiki/2019/7/7a/T--Bilkent-UNAMBG--pzsAg43.png)

**Figure 15 : Fluorescence and Growth Data for pZs sfGFP Ag43**

**DH5α (MG1655) pZs sfGFP Ag43 signifies the DH5α (MG1655) cells having the plasmid pZs sfGFP Ag43. B strain (BL21 DE3) pZs sfGFP Ag43 signifies the B strain (BL21 DE3) having the plasmid pZa sfGFP Ag43.

![](https://static.igem.org/mediawiki/2019/f/fd/T--Bilkent-UNAMBG--pzaAg43.png)

**Figure 16: Fluorescence and Growth Data for pZa sfGFP Ag43**

![](https://static.igem.org/mediawiki/2019/c/cf/T--Bilkent-UNAMBG--emptycells.png)

**Figure 17: Fluorescence and Growth Data for empty cells (without plasmid)**

![](https://static.igem.org/mediawiki/2019/6/68/T--Bilkent-UNAMBG--pzsOD.png)

**Figure 18: Growth Curve for pZs sfGFP Ag43**

![](https://static.igem.org/mediawiki/2019/5/5f/T--Bilkent-UNAMBG--pzaod.png)

**Figure 19: Growth Curve for pZa sfGFP Ag43**

![](https://static.igem.org/mediawiki/2019/f/f8/T--Bilkent-UNAMBG--emptyOD.png)

**Figure 20: Growth Curve of empty cells (i. e. wild type cells)**

# **INDUCTION EXPERIMENTS**

### **Medium Choosing**

In induction experiments, firstly, we tried to find an optimum medium for the cleavage of our single chain insulin (SCI) analogs by TEV protease. We performed induction experiments in intestine like buffer (ILB), TEV reaction buffer (TRB) and MOPS.

Our constructs pZs sfGFP Ag43 and pZe pBAD TEV Ag43 plasmids were transformed to B strain (BL21 Marionette) cells. It was expected that both sfGFP and TEV will be displayed on the cell surface since they are fused with Ag43 cassette. After surface display, TEV protease was expected to cleave sfGFP. Thus, we expected the supernatant ( after centrifugation of cells) to be fluorescent.
(sfGFP Ag43 is under constitutive promoter. TEV Ag43 is under L-arabinose inducible promoter.)
![](https://static.igem.org/mediawiki/2019/1/18/T--Bilkent-UNAMBG--supernatant.png)

**Figure 21: Fluorescence Measurement of The Supernatant**

ara(+) means cells were induced with arabinose. ara(-) means cells were not induced with arabinose. ILB, TRB and MOPS are media.

As seen in Figure 21, TEV cleavage occurred best in MOPS, compared to other media. The fluorescence data regarding TRB, suggests that there might be an experimental error since uninduced sample have a higher fluorescence than the induced one. Considering, the negativity of fluorescence data regarding induced sample in ILB, we thought that sfGFP may not be cleaved. Alternatively, sfGFP may not be expressed. Thus, we decided to be sure of the expression. In other words, we wanted to figure out what the problem is. Is it unsuccessful TEV cleavage or unsuccessful expression ?

Therefore, we measured the fluorescence of cells this time (i.e. the pellet), not the supernatant. If expression occurs successfully, we expected to obtain a high fluorescence data for uninduced samples. We expect to measure a very low fluorescence for induced samples since we measure the fluorescence of the pellet.

Figure 21 is the result of this experiment, namely the fluorescence measurement of the cells. The data satisfied our expectations. Thus, we concluded that the sfGFP is expressed successfully and there is a problem with TEV cleavage in ILB and TRB. The problem may be either related to unsuccessful induction or only unsuccessful cleavage.

![](https://static.igem.org/mediawiki/2019/c/c1/T--Bilkent-UNAMBG--CELL.png)

**Figure 22: Fluorescence Measurement of The Cells**

### **External TEV Cleavage**

(External TEV Cleavage is not an induction experiment. However, we placed this here because of its context.)

In order to understand and solve the problems we experienced regarding TEV cleavage, we decided to perform external TEV cleavage. In external TEV cleavage experiment, pure TEV protease enzyme was added to reaction medium including cells which have only sfGFP Ag43 expressing plasmid. The cell strain used is B strain (BL21 Marionette).

When TEV protease is added into the medium, sfGFP is cleaved. The supernatant fluorescence was measured with the M5 spectrophotometer.

The measurement results are shown in Figure 23. The results were not as we expected, there was an obvious increase in fluorescence when TEV protease was added but this amount was rather insignificant. Thus, we concluded that unsuccessful TEV cleavage is not related to expression of TEV protease by cell. There might be a problem related to TEV recognition site of sfGFP Ag43 cassette. However, the data in Figure shows that TEV cleavage occurs best in intestine like buffer, since the difference between TEV protease treated and untreated samples are higher in intestine like buffer. Hence, this result suggests that intestine medium is appropriate for TEV cleavage.

![](https://static.igem.org/mediawiki/2019/1/10/T--Bilkent-UNAMBG--externaltev.png)

**Figure 23: External TEV Cleavage**

(+) indicates that pure TEV protease was added to the medium, (-) indicates that there is no addition of pure TEV protease.

In addition, if we are to compare the heat release and TEV protease experiments, there is a distinct difference between the positive and negative samples. This also supports that the TEV protease doesn’t cleave the sfGFP. There could also have been a contamination issue with TEV protease sample. The heat release data suggests that our expression system works but there were some complications with the TEV cleavage.

### **Induction & Kinetic Measurement in MOPS**

We also performed kinetic measurements of TEV cleavage. We performed the experiment according to our kinetic measurement protocol. MOPS media was chosen since it is the only medium that provides appropriate conditions both for TEV cleavage and cell growth. TEV reaction buffer and intestine like buffer does not support cell growth.

The construct names given on top of the graphs are the plasmids that the cells have. The cells are B strain (BL21 Marionette).

ara(+) means that the cells were induced with L-arabinose.
ara(-) means that the cells were not induced with L-arabinose.

Figure 24, 25, 26 and 27 show the results of our induction experiment performed on 04.10.2019.
Figure 28, 29, 30 and 31 show the results of our induction experiment performed on 17.10.2019.
Figure 32, 33, 34 and 35 show the results of our induction experiment performed on 20.10.2019.
Considering both graphs, it is seen that there is no significant difference between arabinose treated and arabinose untreated cells.

![](https://static.igem.org/mediawiki/2019/3/3e/T--Bilkent-UNAMBG--a%2Bs.png)

**Figure 24 : Kinetic Measurement after Induction (I)**

![](https://static.igem.org/mediawiki/2019/7/76/T--Bilkent-UNAMBG--s%2Ba.png)

**Figure 25: Kinetic Measurement after Induction (II)**

![](https://static.igem.org/mediawiki/2019/c/c4/T--Bilkent-UNAMBG--s%2Be.png)

**Figure 26: Kinetic Measurement after Induction (III)**

![](https://static.igem.org/mediawiki/2019/0/03/T--Bilkent-UNAMBG--a%2Be.png)

**Figure 27: Kinetic Measurement After Induction (IV)**

![](https://static.igem.org/mediawiki/2019/0/00/T--Bilkent-UNAMBG--s%2Ba1.png)

**Figure 28: Kinetic Measurement after Induction (I)**

![](https://static.igem.org/mediawiki/2019/3/3f/T--Bilkent-UNAMBG--s%2Be2.png)

**Figure 29: Kinetic Measurement after Induction (II)

![](https://static.igem.org/mediawiki/2019/2/25/T--Bilkent-UNAMBG--a%2Be3.png)

**Figure 30: Kinetic Measurement after Induction (III)**

![](https://static.igem.org/mediawiki/2019/f/fd/T--Bilkent-UNAMBG--a%2Bs2.png)

**Figure 31: Kinetic Measurement after Induction**

![](https://static.igem.org/mediawiki/2019/6/60/T--Bilkent-UNAMBG--s%2Be3.png)

**Figure 32: Kinetic Measurement after Induction (I)**

![](https://static.igem.org/mediawiki/2019/9/94/T--Bilkent-UNAMBG--s%2Ba3.png)

**Figure 33: Kinetic Measurement after Induction (II)**

![](https://static.igem.org/mediawiki/2019/6/61/T--Bilkent-UNAMBG--a%2Bs3.png)

**Figure 34: Kinetic Measurement after Induction (III)**

![](https://static.igem.org/mediawiki/2019/5/50/T--Bilkent-UNAMBG--a%2Be.3.png)

**Figure 35: Kinetic Measurement after Induction (IV)**

The results were not quite expected but after reviewing our modelling, we saw that in order the cells with pZA backbone to display the protein, it requires 3 days of incubation. Therefore, we have to adapt to modelling and remake the experiment according to it.

### **Double Induction Experiment in MOPS**

![](https://static.igem.org/mediawiki/2019/d/d6/T--Bilkent-UNAMBG--DI.png)

**Figure 36: Kinetic Measurement after Double Induction**

B Strain (BL21 Marionette) was used.

While practising induction experiments with L-arabinose, we also performed a double induction experiment. The purpose of this experiment was to observe TEV cleavage in a double inducible system. In our single inducible system, we have sfGFP Ag43 under the constitutively active promoter proD and TEV Ag43 under L-arabinose inducible promoter. In this double inducible system, we have sfGFP Ag43 under lacI promoter and TEV Ag43 under NTETO promoter. We had two groups of samples. Both groups were induced with IPTG. The group shown with dark blue in Figure 23 was induced with aTc. The group shown with light blue was not induced with aTc. As seen from Figure 36, the difference between, aTc treated and untreated group is higher than the difference between L-arabinose treated and untreated group of our L-arabinose induction experiment. This may suggest that constitutive expression might be causing problems.

### **Western Blot**

All single transformed IDT BL21 cells were induced with IPTG. Western blot was performed following the heat release. All constructs were fused to Ag43. Therefore we were expecting to see bands around 46 kDa.IDT G3 (Rajpal 15 design with SCI57 a-b chains) and IDT G7 (SCI57 with its own linker a-b chains) were found out to be expressed.

![](https://static.igem.org/mediawiki/2019/a/ad/T--Bilkent-UNAMBG--western.png)

### **ICC**

We performed immunocytochemistry (ICC) assay in order to observe the expression of our SCI constructs. Working principle of immunocytochemistry assay is to provide bacterial cultures that are producing His-Tag containing proteins with His-Tag binding antibodies and visualize the cells under fluorescence microscopy. With primary antibodies, we bind the His-Tag that is expressed with the SCI that is secreted to the cell surface. After primary antibodies, secondary antibodies are used in order to label the primary antibodies with fluorescence activity. Under fluorescence microscopy these cells are visualised.

![](https://static.igem.org/mediawiki/2019/4/43/T--Bilkent-UNAMBG--ICC3.jpg)

ICC Data of IDT-3**

![](https://static.igem.org/mediawiki/2019/9/93/T--Bilkent-UNAMBG--ICC7.jpg)

**ICC Data of IDT-7**

### **Measuring Insulin Activity**

We aimed to perform qPCR on several genes that are upregulated when the insulin receptor is bound. Unfortunately the results are going to be delayed so we are not able to share them on the wiki.

### **References**

**1)**Van der Woude, M. W. and Henderson, I. R. (2008) Regulation and function of Ag43 (flu). Annu. Rev. Microbiol. 62, 153, DOI: 10.1146/annurev.micro.62.081307.162938

**2)**Kjærgaard, K., Hasman, H., Schembri, M. A., and Klemm, P. (2002) Antigen 43-mediated autotransporter display, a versatile bacterial cell surface presentation system. Journal of bacteriology 184, 4197– 4204, DOI: 10.1128/JB.184.15.4197-4204.2002**

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