iGEM 2018 — Team Utrecht

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# Team:Utrecht/Notebook

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Receptor Assay
BRET Assay
Methylation Assay

# Receptor Assay

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August

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September

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October

# BRET Assay

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July

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August

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September

❮

October

# Methylation Assay

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July

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August

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September

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October

Mo

Tu

We

Th

Fr

Sa

Su

### Morning

Performed Calibration 1, 2, and 3 according to protocol. All measurements were performed using the plate reader of Seino.

Table 1. content 96 wells plate cal.1 and cal.2

|  | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| A | L | dd |  |  |  |  |  |  |  |  |  |  |
| B | L | dd |  |  |  |  |  |  |  |  |  |  |
| C | L | dd |  |  |  |  |  |  |  |  |  |  |
| D | L | dd |  |  |  |  |  |  |  |  |  |  |
| E | MS | dd | dd | dd | dd | dd | dd | dd | dd | dd | dd | dd |
| F | MS | dd | dd | dd | dd | dd | dd | dd | dd | dd | dd | dd |
| G | MS | dd | dd | dd | dd | dd | dd | dd | dd | dd | dd | dd |
| H | MS | dd | dd | dd | dd | dd | dd | dd | dd | dd | dd | dd |

##### Calibration 1

* L= 100 uL Ludox CL-X (stored at 4C)
* dd= 100 uL ddH20
* Measurement: Abs600, turn off pathlength correction

##### Calibration 2

* MS= 200 ul Microsphere Stock Solution
* dd= 100 uL ddH20
* green= serial dilution was performed with a micropipet from E1,F1,G1,H1 - E11,F11,G11,H11 by a volume of 100uL. Before every transfer solution was pipetted up and down 3x, after every transfer tips were discharged.
* Measurement: Abs600, re-mix befor putting in plate reader and prevent bubbles, path length correction off

##### Calibration 3

* 1xFC= 200 mL 1xFC (100uL 10x fluorescein + 900ul 1x PBS pH 7.4, tube was covered with foil
* P= 100 uL 1x PBS pH 7.4
* green= serial dilution was performed with a micropipet from A1,B1,C1,D1 - A11,B11,C11,D11 by a volume of 100uL. Before every transfer solution was pipetted up and down 3x, after every transfer tips were discharged.
* Measurement: FL, 530nm/30nm bandpass, 25-30nm with recommened excitation of 485nm, emission 520-530nm of the filter. Path length correction was turned off

Table 2. content 96 wells plate cal.3

|  | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| A | 1xFC | P | P | P | P | P | P | P | P | P | P | P |
| B | 1xFC | P | P | P | P | P | P | P | P | P | P | P |
| C | 1xFC | P | P | P | P | P | P | P | P | P | P | P |
| D | 1xFC | P | P | P | P | P | P | P | P | P | P | P |
| E |  |  |  |  |  |  |  |  |  |  |  |
| F |  |  |  |  |  |  |  |  |  |  |  |
| G |  |  |  |  |  |  |  |  |  |  |  |
| H |  |  |  |  |  |  |  |  |  |  |  |

### Afternoon

##### LBC plates were made according to the protocol used on the wall

* 250ml LB 2x added to melted 250 ml WA 2x using a microwave
* 0.5ml was added to final solution
* plates were dried in 37C incubator

##### Transformation device 3 + negative control interlab study

* Device 3 (number 5) showed a low GFP expression, so it was tried to re-preform the tranformation. Negative control of the interlab (number 1) was not performed last time due to lack of LBC plates so was also performed.
* [Protocol Transformation](https://2018.igem.org/Team%3AUtrecht/Protocol#Transformation)

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## 21/08/18

**Lorenzo**

1/9M (RFP) was transformed from the iGEM kit according to the protocol. The coding sequence of the custom receptor, and copper promoter flanked by biobrick sites were ordered from IDT and used directly.

## 22/08/18

**Lorenzo**

Transformation was successful and a colony was picked and inoculated in chloramphenicol.

## 23/08/18

**Felix, Lorenzo**

Miniprep was performed according to the [protocol.](https://2018.igem.org/Team%3AUtrecht/Protocol#PlasmidPurification)
Cu-promotor → 250 ng/μl
Custom receptor → 360 ng/μl
RFP → 91.7 ng/μl

Cu-promoter and RFP were placed in pSB1K3, custom receptor was placed in pSB1K3 as well. Cu-promoter DNA was digested with EcoRI and SpeI, RFP DNA was digested with XbaI and PstI. The custom receptor and the target-vector were digested with EcoRI and PstI.

For each of the three samples a mastermix was made. For each digestion the DNA concentration was reduced to 25 ng/ul. 4 ul of DNA sample and target vector sample were transferred to a PCR tube with 4 ul of the according restriction mix. The samples were incubated for 1 hour at 37 C. After 1 hour the samples were taken out of the incubator and enzymes were heat deactivated at 80°C for 20 min. 1ul of target vector (pSB1K3), 2 ul of Cu-promotor restricted DNA, and 2 ul restricted RFP DNA were added to one PCR tube. Furthermore 1ul DNA T4 ligase buffer and 0.5 ul of T4 ligase were added. The total volume was made up to 10 ul with 3.5 ul dH2O. The samples were incubated for one hour (RT). Enzymes were deactivated with heat kill 80°C 20 min.

DNA was transformed in DH5α according to the iGEM protocol.

## 24/08/18

**Lorenzo**

The transformation was successful. 8 colonies were picked to perform colony PCR according to the [protocol](https://2018.igem.org/Team%3AUtrecht/Protocols#ColonyPCR). (Cu-promotor::RFP 100%, Cu-promotor::RFP 500%, Custom receptor 100%, Custom Receptor 500%).

![](https://static.igem.org/mediawiki/2018/6/69/T--Utrecht--2018-RA24_1-Notebook.jpg)
![](https://static.igem.org/mediawiki/2018/2/2a/T--Utrecht--2018-RA24-2-Notebook.jpg)
![](https://static.igem.org/mediawiki/2018/f/f1/T--Utrecht--2018-RA24-3-Notebook.jpg)
![](https://static.igem.org/mediawiki/2018/8/85/T--Utrecht--2018-RA24-4-Notebook.jpg)

Note: The date on the plates is date of preparation not transformation

## 27/08/18

**Felix**

Samples from colony PCR (24/08/18) were analysed with a 0.8% Agar gel electrophoresis.
Custom receptor = 1926 bp
Cu-promoter::RFP = 1238 bp

![](https://static.igem.org/mediawiki/2018/e/ec/T--Utrecht--2018-R827f1-Notebook.jpg)

|  |  |
| --- | --- |
| 1-8 | Custom receptor 100% |
| 9-16 | Custom receptor 500% |
| 17-24 | Cu::RFP 100% |
| 25-32 | Cu::RFP 500% |

## 28/08/18

**Lorenzo, Felix**

The results obtained in the gel electrophoresis DNA analysis (27/8/18) was not satisfactory. Therefore four new colonies were picked for colony PCR in accordance with the [protocol](https://2018.igem.org/Team%3AUtrecht/Protocols#ColonyPCR).

|  |  |
| --- | --- |
| 1-4 | Cu::RFP 100% |
| 5-8 | Cu::RFP 500% |
| 9-12 | Custom receptor 100% |
| 13-16 | Custom receptor 500% |

The PCR samples were analysed with gel electrophoresis using 0.8% agarose gel.
Custom receptor = 1926 bp
Cu-promoter::RFP = 1238 bp

![](https://static.igem.org/mediawiki/2018/5/5e/T--Utrecht--2018-RA28-Notebook.jpg)

Samples 4, 9, 12, 13, 14 were selected and inoculated in 5 ml LB containing kanamycin overnight. A safe of these samples was made as well.

As the Cu::RFP sample did not yield the desired results, new cloning procedure was initiated using the iGEM protocol and buffer 2.1 instead of enzyme specific buffers. In order to ensure that we would have enough genetic material, we cloned the gBlock `copper-promoter blunt into the vector pBSK.

## 29/08/18

**Felix**

The DNA of the (28/08/18) samples was purified according to  [the miniprep protocol](https://2018.igem.org/Team%3AUtrecht/Protocols#PlasmidPurification).

| Sample | Concentration ng/µl | Description |
| --- | --- | --- |
| 1 - 8 | 69.9 | Cu-Promotor+ RFP |
| 9 | 77.5 | Custom Receptor |
| 12 | 91.8 | Custom Receptor |
| 13 | 49.4 | Custom Receptor |
| 14 | 85.4 | Custom Receptor |

The prior prepared clonation (28/08/18) was transformed in DH5α according to the iGEM protocol.

Safe made from samples Samples 4, 9, 12, 13, 14 (08/28/18)

![](https://static.igem.org/mediawiki/2018/2/2c/T--Utrecht--2018-RA29-Notebook.jpg)

## 30/08/18

**Felix**

No colonies of the transformation(29/8/18) were observed.
The purified DNA of samples 4,12,14 (29/8/18) were send for sequencing with forward and reverse primer.

![](https://static.igem.org/mediawiki/2018/f/f4/T--Utrecht--2018-RA30-Notebook.jpg)

## 05/09/18

**Lorenzo**

Samples from sequencing were analysed. Custom receptor (12) was placed in the pSB1K3 backbone correctly. The sequence of Cu-promoter::RFP was difficult to interpret. Therefore Cu-promoter::RFP (4) was incubated again in 5 ml LB + Kanamycin from the safe overnight.

## 06/09/18

**Jolijn**

The Cu::RFP DNA was purified according to the [miniprep protocol](https://2018.igem.org/Team%3AUtrecht/Protocols#PlasmidPurification) with 5 ml bacteria instead of 1.5 ml and eluted in 20 µl elution buffer. DNA was sent for sequencing.

| Cu + RFP (4) Code: | Primer |
| --- | --- |
| 1BA9ZAB414 | VF |
| 1BA9ZAB413 | VR |

## 10/09/18

**Lorenzo**

The sequence of Cu::RFP did not contain the copper promotor, only RFP.

## 11/09/18

**Lorenzo**

The RFP DNA was cloned into the Copper-promoter vector by cutting the copper-promoter vector with SpeI and PstI, and the RFP vector with XbaI and PstI overnight at 37 °C.

## 12/09/18

**Felix**

The prior cut samples were ligated. 2.4 µl RFP DNA was used as insert with 1 µl of Cu promotor backbone. After heat inactivation the sample was transformed according to the iGEM protocol in DH5α and plated on Ampicillin plates.

## 13/09/18

**Lorenzo**

Transformation was successful. However, close examination of the samples under the fluorescence binoculars revealed that none of the colonies expressed RFP.

![](https://static.igem.org/mediawiki/2018/c/c9/T--Utrecht--2018-RS13-Notebook.jpg)

2 colonies were picked and inoculated in 5 ml LB + ampicillin.

## 22/09/18

**Lorenzo**

Since our previous attempts didn’t yield any results we decided to place the copper promoter gBlock with the restriction enzymes EcoRI and SpeI. Simultaneously, the RFP vector (PSB1C3) was cut with EcoRI and XbaI. Both reactions were done overnight in NEB Buffer 2.1.

## 23/09/18

**Lorenzo**

The digestion product from 09/22/18 was ligated according to the iGEM ligation protocol. 2 µl of ligation product was transformed to *E. coli* DH5α.

## 24/09/18

**Lorenzo**

The reaction mix was inactivated by heating it to 80°C for 20 minutes. A ligation was set up with the fragments, and 9 µl of the ligation product was transformed to 100 µl of competent DH5α.

## 25/09/18

**Felix**

The transformation was successful. 6 colonies could be spotted. These were inoculated overnight in 5 ml LB with chloramphenicol.

## 26/09/18

**Lorenzo, Felix**

The DNA of the inoculated cultures was minipreped according to the [miniprep protocol](https://2018.igem.org/Team%3AUtrecht/Protocols#PlasmidPurification). After the first spin down we noticed that the bacterial pellet of one of the colonie number 6 was slightly red. Because it is known that RFP expressing bacteria turn red even without excitation, we concluded that it was most likely that this colony expressed RFP, and thus that the transformation worked. After the isolation of the DNA, a colony PCR was done with the VF and VR primer. The total size of the band that we expected was 824 + 156 -6 = 974 bp for the fragment + 271 bp for the part outside the BB\_sites generated by the VF and VR primer. The total size of the fragment should thus be 1245 bp. The fragment in lane 6 lies between the 1000 bp band and the 1500 bp band and starts slightly lower than the middle, indicating that it is most likely a band of correct size.

The DNA of this colony was send for sequencing.

![](https://static.igem.org/mediawiki/2018/7/7a/T--Utrecht--2018-RS27-Notebook.jpg)

## 29/09/18

**Lorenzo**

The sequencing results showed that the construct was correct. Since both the promoter::RFP fusion, and the Custom receptor were now available to us on plasmid backbones with different resistance genes, both constructs were transformed to  *E. coli*  DH5α.

![](https://static.igem.org/mediawiki/2018/b/b9/T--Utrecht--2018-RS29-Notebook.jpg)

## 30/09/18

**Lorenzo, Felix**

The transformation was successful.
A stock solution of 50 mM CuSO4 was made for experiments with the newly transformed copper sensitive bacteria. 2 colonies were inoculated overnight for further experiments.

![](https://static.igem.org/mediawiki/2018/8/8b/T--Utrecht--2018-RS30-Notebook.jpg)

## 01/10/18

**Lorenzo**

100 µl of the overnight cultures from 30/09/18 was transferred to two tubes containing 5 ml of clean LB. One tube contained 500 µM CuSO4. Both tubes were inoculated for 45 minutes at 37°C.
400 µl of this culture was washed in 1 ml of PBS and RFP expression was measured with an emission scan between 570 and 640 nm in a Carry Eclipse Fluorescence Photospectrometer using 400 µl of the copper bacterium containing PBS solution. The excitation wavelength used was 555 nm. No fluorescence could be measured, which was probably due to the low amount of bacteria.

![](https://static.igem.org/mediawiki/2018/c/c8/T--Utrecht--2018-RO1-Notebook.jpg)

## 02/10/18

**Jolijn**

A new overnight inoculation of the double transformed bacteria was made.

## 03/10/18

**Felix, Lorenzo**

The experiment of 10/01/18 was repeated, but this time CuSO4 was added to the inoculation at a final concentration of 500 µM, 250 µM, 50 µM, and 5 µM, or 0 µM. The Bacteria were inoculated for 2 hrs at 37°C.

## 12/10/18

**Felix**

The following bacteria (DH5α) were inoculated in 5 ml minimal medium, and 5 ml LB with matching antibiotic.

Cu-promotor::RFP // Custom receptor → Ampicillin, Chloramphenicol
Cu-promotor::RFP → Chloramphenicol

## 13/10/18

**Jolijn, Felix, Lorenzo**

Bacteria in minimal medium did not grow. Cu-promoter::RFP // Custom receptor samples were red coloured while bacteria not containing the custom receptor were yellowish. Fluorescence images were taken to confirm RFP expression. The bacteria containing the Cu-promoter::RFP // Custom receptor were washed 1x in M9 and incubated again overnight in M9 containing 0.4% glucose.

In order to confirm aspartic acid induced RFP expression the experiment was repeated. The following bacteria (DH5α) were inoculated in 5 ml M9 + 0.4% glucose, and in 5 ml LB with matching antibiotic.

Cu-promotor::RFP // Custom receptor → Ampicillin, Chloramphenicol
Cu-promotor::RFP → Chloramphenicol

![](https://static.igem.org/mediawiki/2018/a/a8/T--Utrecht--2018-RO13-1-Notebook.jpg)

Cu-promoter :: RFP Normarski

![](https://static.igem.org/mediawiki/2018/9/94/T--Utrecht--2018-RO13-2-Notebook.png)

Cu-promoter :: RFP Nomarski Fluorescence

![](https://static.igem.org/mediawiki/2018/3/3d/T--Utrecht--2018-RO13-3-Notebook.png)

Cu-promoter :: RFP // Custom receptor

![](https://static.igem.org/mediawiki/2018/e/e8/T--Utrecht--2018-RO13-4-Notebook.png)

Cu-promoter::RFP // Custom receptor fluorescence (falsely coloured)

## 14/10/18

**Felix, Jolijn**

Bacteria in M9 did not grow. Bacteria in LB did grow but did not express GFP observed with the naked eye. 4 ml bacteria of each of the two samples was washed with 1x with M9 and incubated with M9 + 0.4% glucose.

After 4 hours of incubation in M9 + 0.4% glucose the samples were split up in the following fractions. After 4 hours 500 μl was used for fluorescence measurements using the prior described fluorescence photospectrometer (ex. 585 nm). After 7 hours the remaining 500 μl was used for measurements.

| Sample |  | Asp (10 μ 50 mM) | Cu (2 μl 25 mM) |
| --- | --- | --- | --- |
| Custom Receptor | 1 | + | - |
| Custom Receptor | 2 | - | - |
| Cu-promoter :: RFP | 3 | + | - |
| Cu-Promoter :: RFP | 4 | - | + |
| Cu-Promoter :: RFP | 5 | - | - |

![](https://static.igem.org/mediawiki/2018/b/b4/T--Utrecht--2018-RO14-00-Notebook.png)
![](https://static.igem.org/mediawiki/2018/5/55/T--Utrecht--2018-RO14-0-Notebook.png)

Sample 1 is not representative at 8 hours since the remaining volume of bacteria was less than the volume in the cuvette. Therefore this sample was diluted which resulted in a increased intensity as the cuvette was now fully filled, however, the measurement did not reach its full potential.

The Cu-promoter::RFP // Custom receptor sample washed (13/10/18) and incubated overnight was split up in two 4 ml fractions. To one fraction 40 μl 50 mM Aspartic acid was added and incubated for 4 hours. After 4 hours 500 μl was used for fluorescence measurements, this was repeated at 8 and 11 hours

Although these data are not conclusive it indicates that the custom receptor has a reaction on the addition of aspartate. Therefore new samples were inoculated in 5 ml LB.

|  |  |
| --- | --- |
| Custom // Cu-promoter :: RFP | 3x |
| Custom // Cu-promoter :: RFP Asp+ 500 μM | 1x |
| Cu-promoter :: RFP | 3x |
| Cu-promoter :: RFP Asp+ 500 μM | 1x |
| Cu-promoter :: RFP 50 μM CuSO4 | 1x |

![](https://static.igem.org/mediawiki/2018/1/1b/T--Utrecht--2018-RO14-1-Notebook.png)
![](https://static.igem.org/mediawiki/2018/7/7f/T--Utrecht--2018-RO14-2-Notebook.png)

## 15/10/18

**Felix, Jolijn**

The three custom receptor samples and one co-promoter::RFP sample did not fully grow after 9 hours of inoculation, these samples were put back into the incubator for 15 hours and can be seen in the table underneath. The other samples were incubated for 4 hours in M9.

| Sample | Volume | OD600 |
| --- | --- | --- |
| 1. Co-promotor + RFP | 4 mL | 1.54 |
| 2. Co-promotor + RFP | 4 mL | 1.35 |
| 3. Co-promotor + RFP | 3 mL | 2.70 |
| 4. Promotor + RFP + Aspartate | 3 mL | 0.47 |

Copper was added to the samples incubated in M9 in concentrations of 25 and 50 µM. RFP expression was measured at t = 0, t = 2. However, results from the scan were inconclusive.

The three custom receptor samples and one cu-promoter::RFP sample incubated in total for 24 hours. These samples were used for new inoculations as described below in increased volumes. The remaining volume of the inoculations were pelleted and washed once with Milli Q and resuspended in M9, in total, all samples were resuspended in 5 ml M9 (Orange in the table). During the pelleting the pellets of samples containing custom receptor turned red.

| Sample | Medium | Volume |
| --- | --- | --- |
| Custom receptor // Cu-promoter::RFP | LB + ampicillin and chloramphenicol | 100 ml |
| Cu-promoter::RFP | LB + chloramphenicol | 100 ml |
| Custom receptor // Cu-promoter::RFP | SV + 0.4% glucose + ampicillin and chloramphenicol | 100 ml |
| Cu-promoter::RFP | SV + 0.4% glucose + chloramphenicol | 100 ml |
| Cu-promoter::RFP | LB + chloramphenicol | 5 ml |
| Cu-promoter::RFP | LB + 25 μM CuSO4 + chloramphenicol | 5 ml |
| Cu-promoter::RFP | LB + 50 μM CuSO4 + chloramphenicol | 5 ml |
| Cu-promoter::RFP | LB + 100 μM CuSO4 + chloramphenicol | 5 ml |
| 3x Custom receptor // Cu-promoter::RFP | M9 + 0.4% glucose | 5 ml |
| 1x Cu-promoter::RFP | M9 + 0.4% glucose | 5 ml |

![](https://static.igem.org/mediawiki/2018/0/00/T--Utrecht--2018-RO15-Notebook.jpg)

## 16/10/18

**Felix, Jolijn**

The samples that were incubated with copper the day before, were measured in PBS with 0.4% glucose.
Copper was added to the samples with custom receptor//Cu-promotor::RFP and Cu-promotor::RFP in concentrations of 25, 50, 100 and 250 µM. Next, RFP fluorescence was measured at t = 0h, t = 1.5h and t = 2.5h.

![](https://static.igem.org/mediawiki/2018/7/71/T--Utrecht--2018-RO16-Notebook.jpg)
![](https://static.igem.org/mediawiki/2018/e/e9/T--Utrecht--2018-RO16-2-Notebook.jpg)

5 mL of the 25 mL overnight LB cultures of Custom receptor // Cu-promoter::RFP (OD600 of 0.675) and Cu-promoter::RFP (OD600 of 0.690) were used to attain the following solutions:

1. Sample with nothing added
2. Sample with 125 µM aspartate
3. Sample with 250 µM aspartate
4. Sample with 500 µM aspartate
5. Sample with 50 µM copper

RFP fluorescence was measured at t = 0, t = 4, and t = 6. There was no fluorescence. Upon measuring the fluorescence of the strain from which the samples originated (incubated in LB overnight), only slight fluorescence was visible.

## 30/07/18

**Lorenzo, Floor**

Isolated biobricks (listed in table below) and transformed them to *E. coli* Dh5ɑ according to the transformation protocol.

| Biobrick | Kit location\* | Part name | Part size (bp) |
| --- | --- | --- | --- |
| BBa\_K608003 | 1/5A | Promoter +RBS | 56 |
| BBa\_K569017 | 1/8K | CheY | 390 |
| BBa\_K629003 | 1/18G | CheZ | 644 |
| BBa\_I759001 | 3/3J | Rluc | 936 |
| BBa\_B0017 | 3/6C | Terminator | 128 \*\* |
| BBa\_I15017 | 4/21N | eYFP | 717 |

\* location name is as follows: plate number/well number (e.g. 1/5A means plate 1 well 5A)
\*\* this part contains two copies of BBa\_B0010 (64 bp each) but the exact size of the fragment is unspecified on the wiki page.

## 31/07/18

**Lorenzo, Floor**

Transformation was successful, colonies were picked and inoculated in 5 ml LB containing the correct antibiotic.

## 01/08/18

**Lorenzo, Floor**

Isolated the plasmids containing the biobricks from the inoculated colonies from 31/07/18 according to the [plasmid purification protocol](https://2018.igem.org/Team%3AUtrecht/Protocols#PlasmidPurification). (for concentrations see table below)

|  |  |  | Concentration (ug/ml) |  |
| --- | --- | --- | --- | --- |
| Biobrick | Kit location\* | Part name | Replicate 1 | Replicate 2 |
| BBa\_K608003 | 1/5A | Promoter +RBS | 76,8 | 77,8 |
| BBa\_K569017 | 1/8K | CheY | 51 | 50,5 |
| BBa\_K629003 | 1/18G | CheZ | 83,1 | 111,9 |
| BBa\_I759001 | 3/3J | Rluc | 81,2 | 89,7 |
| BBa\_B0017 | 3/6C | Terminator | 137,8 | 75,4 |
| BBa\_I15017 | 4/21N | eYFP | 70 | 70,9 |

Performed a PCR, as described in the [PCR protocol](https://2018.igem.org/Team%3AUtrecht/Protocols#PCR), to amplify the parts CheY, CheZ, Rluc, eYFP so that they can be used for Gibson assembly later in the project. The primers used are listed in the table below.

| Part | Primer name | Primer sequence 5’--> 3’ |
| --- | --- | --- |
| CheZ | CheZ\_F1 | gccagtgaattgtaatacgactcactatagggcgaattgggaattcgcggccgcttctag |
|  | CheZ\_R1 | CCTTGGAAGCCATTCCACCTCCACCTCCAAATCCAAGACTATCCAACAAATCG |
| Rluc | Rluc\_F1 | AGTCTTGGATTTGGAGGTGGAGGTGGAATGGCTTCCAAGGTGTACG |
|  | Rluc\_R1 | tcactaaagggaacaaaagctggagctccaccgcggtggcctgcagcggccgctactag |
| eYFP | YFP\_F1 | gccagtgaattgtaatacgactcactatagggcgaattgggaattcgcggccgcttctag |
|  | YFP\_R1 | CTTATCAGCCATTCCACCTCCACCTCCCTTGTACAGCTCGTCCATGCCG |
| CheY | CheY\_F1 | CGAGCTGTACAAGGGAGGTGGAGGTGGAATGGCTGATAAGGAATTGAAG |
|  | CheY\_R1 | tcactaaagggaacaaaagctggagctccaccgcggtggcctgcagcggccgctactag |

## 02/08/18

**Lorenzo**

Performed a gel electrophoresis on the PCR product from 01/08/18 on a 1.5% agarose gel according to the [gel electrophoresis protocol](https://2018.igem.org/Team%3AUtrecht/Protocols#GelElectrophoresis). The resulting bands were isolated according to the [gel purification protocol](https://2018.igem.org/Team%3AUtrecht/Protocols#GelExtraction).

![](https://static.igem.org/mediawiki/2018/3/3e/T--Utrecht--2018-BA2-figure1.jpg)

| PCR product | Concentration (ug/ml) |
| --- | --- |
| CheY | / |
| Rluc | 45,6 |
| CheZ | 59,9 |
| eYFP | 26,6 |

A second PCR was performed to retrieve CheY. Also eYFP, LuxA, and LuxB were amplified with overhang extension PCR to add a gibson overhang to these parts. Used primers are listed in the table below.

| Part | Primer name | Sequence 5’ -> 3’ |
| --- | --- | --- |
| LuxA | M13 fwd | tgtaaaacgacggccagt |
|  | LuxAB\_Gibson\_R1 | gatttGGAGGTGGAGGTGGAATGAAGTTTGGAAACTTCCTG |
| LuxB | LuxAB\_Gibson\_F1 | gatttGGAGGTGGAGGTGGAATGAAGTTTGGAAACTTCCTG |
|  | T3 | tccctttagtgagggttaat |
| eYFP | YFP\_F1 | gccagtgaattgtaatacgactcactatagggcgaattgggaattcgcggccgcttctag |
|  | YFP\_R1 | CTTATCAGCCATTCCACCTCCACCTCCCTTGTACAGCTCGTCCATGCCG |
| CheY | CheY\_F1 | CGAGCTGTACAAGGGAGGTGGAGGTGGAATGGCTGATAAGGAATTGAAG |
|  | CheY\_R1 | tcactaaagggaacaaaagctggagctccaccgcggtggcctgcagcggccgctactag |

The PCR product was ran on a 1.5% agarose gel (figure 2)

![](https://static.igem.org/mediawiki/2018/1/1a/T--Utrecht--2018-BA2-figure2.jpg)

## 03/08/18

**Lorenzo**

LUXA and LuxB were isolated using PCR directly from the gBlock, since we expected the product of 02/08/18 to contain an error. The PCR product was ran on a 0.8% agarose gel and the correct bands were isolated according to the [gel extraction protocol.](https://2018.igem.org/Team%3AUtrecht/Protocols#GelExtraction)

![](https://static.igem.org/mediawiki/2018/e/e1/T--Utrecht--2018-BA3-Notebook.jpg)

## 06/08/18

**Floor**

The CheZ and RLuc, and LuxA and LuxB fragments were fused together using Gibson assembly as described in the [Gibson protocol](https://2018.igem.org/Team%3AUtrecht/Protocols#Gibson). 9 µl of each Gibson product was transformed to 100 µl of DH5ɑ, since this increases the amount of successful transformations.

## 07/08/18

**Floor**

The transformations were successful. 2 colonies from each plate were picked and analysed for bands of a correct size through colony PCR as described in the [colony PCR protocol](https://2018.igem.org/Team%3AUtrecht/Protocols#ColonyPCR). These bacteria were inoculated overnight at 37 °C in a rotary shaker.

![](https://static.igem.org/mediawiki/2018/f/f3/T--Utrecht--2018-BA7-Notebook.jpg)

## 08/08/18

**Lorenzo**

DNA of inoculations (07/08/18) was purified in accordance with the [miniprep protocol](https://2018.igem.org/Team%3AUtrecht/Protocols#PlasmidPurification). The acquired DNA was sent for sequencing.

## 09/08/18

**Lorenzo**

PCR was performed according to the protocol to create the correct overhangs for gibson assembly of CheY.

## 10/08/18

**Jolijn, Felix**

PCR product from 09/08/18 was ran on 0.8% gel and isolated the DNA using the [Gel purification protocol](https://2018.igem.org/Team%3AUtrecht/Protocol#GelExtraction)

## 15/08/18

**Floor, Lorenzo**

eYFP::CheY was assembled according to the [Gibson cloning protocol](https://2018.igem.org/Team%3AUtrecht/Protocol#Gibson). The cloning product was transformed into DH5α according to the iGEM transformation protocol.

## 16/08/18

**Lorenzo**

Made a plate containing the positive colonies and inoculated them overnight.

Colony PCR of ((18/08/18 eYFP::CheY)(08/07/18 CheZ::Rluc)) was performed according to the [protocol](https://2018.igem.org/Team%3AUtrecht/Protocols#ColonyPCR). Positive samples were selected and inoculated overnight.

## 17/08/18

**Felix**
eYFP::CheY DNA 16/08/18

| Inoculation from colony | Concentration (ng/µl) |
| --- | --- |
| 1 | 224.5 |
| 2 | 277.5 |
| 3 | 334.4 |
| 4 | 280.4 |

## 20/08/18

**Felix**

The purified DNA samples (17/08/18) were sent for sequencing with m13 forward and reverse primer.

## 27/08/18

 **Lorenzo, Floor**

Performed a PCR of CheY, Lux AB, CheZ and eYFP with the following primers:

| DNA | Forward Primer | Reverse Primer |
| --- | --- | --- |
| 1. CheY (kit) | Long Forward | CheY reverse |
| 2. CheY (kit) | Short forward | CheY reverse |
| 3. CheY (Gblock) | GblockCheY forward | Reverse 2 |
| 4. LuxAB | LuxAB forward, CheZ overhang | M13 reverse |
| 5. CheZ (kit) | CheZ forward 1 | CheZ reverse, luxAB overhang |
| 6. eYFP | eYFP forward | eYFP reverse |

The samples were ran on an 0.8% gel and bands were isolated

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| 1 | 2 | 3 | 4 | 5 | 6 |
| 10 | 10.1 | 5.7 | 3.7 | 0.9 | 0 |

![](https://static.igem.org/mediawiki/2018/a/a3/T--Utrecht--2018-BA27-Notebook.png)

## 28/08/18

**Floor**

Repeat of 27/08/18, difference: PCR settings as KOD polymerase with 35 cycles.
Ran the product on 0.8% (w/v) agarose gel and isolated it using the [gel purification protocol](https://2018.igem.org/Team%3AUtrecht/Protocols#GelExtraction).

![](https://static.igem.org/mediawiki/2018/5/59/T--Utrecht--2018-BA28-Notebook.png)

The fragments were used to perform a Gibson assembly of eYFP::CheY, and 9 µl of the product was transformed to 100 µl DH5α.

## 29/08/18

**Floor**

We checked the plates from 28/08/18 but none of the transformations were successful.

## 03/09/18

**Lorenzo**

We performed the gibson reaction of eYFP::CheY again. The gibson product was transformed directly to DH5α. Furthermore, a Gibson overhang for the CheZ and LuxAB fragments was created by PCR with primers that were extended. The PCR products were ran on a 0.8% agarose gel. The gel revealed faint bands, but no ladder. Hence we didn’t proceed with these samples.

## 11/09/18

**Lorenzo**

Since our constructs used during our project needed a promoter in order to be expressed promoter BBa\_K608003 was isolated from the DNA distribution kit (plate 1 well 5A). This DNA was transformed to DH5α.

## 12/09/18

**Felix**

Colonies from 11/09/18 were picked and inoculated overnight.

## 13/09/18

**Lorenzo**

DNA from inoculations was isolated according to the [miniprep protocol](https://2018.igem.org/Team%3AUtrecht/Protocols#PlasmidPurification). The promoter part was was cloned in front of Both eYFP::CheY from 28/08/18, and CheZ::RLuc using the iGEM 3A assembly protocol. The samples were transformed to DH5α.

## 14/09/18

**Lorenzo**

eYFP::CheY plates were observed under the fluorescence binoculars. 2 YFP colonies could be observed. These colonies were inoculated overnight at 37°C.

## 15/09/18

**Felix**

The inoculated bacteria were used for a miniprep and the DNA was send for sequencing.

## 06/10/18

**Felix**
**Oligo annealing**

Oligos containing the promoter CheZ::RLuc part sequence were annealed according to the [annealing protocol](https://2018.igem.org/Team%3AUtrecht/Protocols#Annealing)

**CheZ Rluc Digestion**

Two CheZ::Rluc samples (CheZ::Rluc 440 ng/μl, CheZ::Rluc 430 ng/μl) were digested with Xbal and EcoR1 according to the iGEM restriction digest protocol.

**Oligo CheZ::Rluc ligation**

Protomoter Oligo was ligated into the Xbal Rluc digested CheZ::Rluc backbone according to the iGEM T4 ligase protocol . 1 μl of CheZ::Rluc backbone with 1 μl of oligo was used. Both 440 ng/μl and 430 ng/μl sample were ligated with the oligo.

**Transformation**

The new vector product (promoter::CheZ::Rluc) was transformed into DH5α according to the iGEM transformation protocol.

* CheZ Rluc A
* CheZ Rlux B

Anneal A = Oligo insert::CheZ::Rluc (originating from sample CheZ::Rluc 430 ng/μl )
Anneal B = Oligo insert::Chez::Rluc (originating from sample CheZ::Rluc 440 ng/μl )

## 07/10/18

**Felix**

Transformation was succesful

Three colonies were picked from transformation A and two colonies were picked from transformation B, both were inoculated in 5 ml LB containing ampicillin.

![](https://static.igem.org/mediawiki/2018/5/59/T--Utrecht--2018-BO7-Notebook.png)

## 08/10/18

**Lorenzo**

DNA was purified from the following samples using the [miniprep protocol](https://2018.igem.org/Team%3AUtrecht/Protocols#PlasmidPurification) and sent for sequencing.

| Sample | Concentration ng/μl |
| --- | --- |
| CheZ Rluc A4 | 584.1 |
| CheZ Rluc B4 | 549.6 |
| CheZ Rluc A2 | 522.3 |
| CheZ Rluc A1 | 463.8 |
| CheZ Rluc B3 | 539.9 |

The Gibson cloning was transformed in DH5α according to the [protocol](https://2018.igem.org/Team%3AUtrecht/Protocols#Gibson).

## 09/10/18

**Lorenzo**

Transformation was successful

Two colonies were picked and inoculated in 5 ml LB containing ampicillin.

![](https://static.igem.org/mediawiki/2018/0/00/T--Utrecht--2018-BO9-Notebook.png)

## 10/10/18

**Lorenzo, Felix**

DNA from the two inoculation of 1/5A::CheZ::Rluc after Gibson cloning was retrieved with miniprep according to the [protocol](https://2018.igem.org/Team%3AUtrecht/Protocols#PlasmidPurification) in the following concentrations.

Sample 1: 139.9 ng/μl
Sample 2: 75.5 ng/μl

## 11/10/18

**Felix, Jolijn, Mike**

In order to implement the complete BRET-pair and the mutated TAR-receptor, all three constructs needed to be in different vectors. Therefore, the CheZ Rluc and 1/5A::eYFP::Chey DNA was put in the iGEM pSB1K3 vector. Both samples including the vector were cut with EcoRI and PstI. Assembly of products was performed according to the iGEM 3A assembly protocol resulting in the following samples:

1/5A eYFP CheY in pSB1K3
1/5A CheZ Rluc in pSB1K3 (sample 1)
1/5A CheZ Rluc in pSB1K3 (sample 2)

These samples were transformed in combination with the mutated TAR receptor DNA. The following samples were transformed.

| Sample | Content | Cell line |
| --- | --- | --- |
| 1 | SDM Q491A | DH5α |
| 2 | SDM E309A | DH5α |
| 3 | [⅕A::eYFP::CheY] Kan | U1250 |
|  | [SDM Q491A] Chlo |  |
|  | [CheZ::Rluc] Amp |  |
| 4 | [CheZ::Rluc (sample 1)] Kan | U1250 |
|  | [SDM Q491A] Chlo |  | |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | |  | [⅕A::eYFP::CheY] Amp |  | |  |  |  | | --- | --- | --- | | 5 | [CheZ::Rluc (sample 2)] Kan | U1250 | |  | [SDM Q491A] Chlo |  | |  |  |  | | --- | --- | --- | |  | [⅕::eYFP::CheY] Amp |  | | 6 | [[⅕A::eYFP::CheY] Kan | U1250 | |  | [SDM E309A] Chlo |  | |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | |  | [CheZ::Rluc] Amp |  | |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | 7 | [CheZ::Rluc (sample 1)] Kan | U1250 | |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | |  | [SDM E309A] Chlo |  | |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | |  | [⅕A+eYFP+CheY] Amp |  | |  |  |  | | --- | --- | --- | | 8 | [CheZ::Rluc (sample 2)] Kan | U1250 | |  | [SDM E309A] Chlo |  | |  | [⅕A::eYFP::CheY] Amp |  | |  |  |  |  |  |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | 9 | [⅕A::eYFP::CheY] Kan | DH5α | |  |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | --- | | 10 | [CheZ::Rluc (sample 1)] Kan | DH5α | |  |  |  |  | | --- | --- | --- | --- | | 11 | [CheZ::Rluc (sample 2)] Kan | DH5α |  | | | | | | | | | | | |

## 12/10/18

**Felix, Lorenzo**

SDM transformations (Lab Journal lab journal Methylation) and triple transformations were successful. When the plates were inspected under the fluorescence binoculars, several eYFP positive colonies could be identified that weren’t RFP positive. This indicated that these bacteria contained all 3 plasmids, and weren’t antibiotic resistant due to self ligation events of the linearized vector backbone used on 11/10/18.

2 colonies were picked and inoculated in 5 ml LB with ampicillin, kanamycin, chloramphenicol. The selection of colonies was based on increased YFP expression and low RFP expression (background plasmid signal).

![](https://static.igem.org/mediawiki/2018/a/ad/T--Utrecht--2018-BO12-2-Notebook.png)
![](https://static.igem.org/mediawiki/2018/d/da/T--Utrecht--2018-BO12-Notebook.png)

The following samples were transformed

| Sample | Cell line |
| --- | --- |
| 6/15B (TAR WT), | U1250 |
| FRET pair | U1250 |
| BRET pair (1/5A::CheZ::Rluc)(1/5A::eYFP::CheY) | U1250 |
| 6/15B (TAR WT), FRET pair | U1250 |
| 6/15B (TAR WT), BRET pair (1/5A::CheZ::Rluc)(1/5A::eYFP::CheY) |
U1250 | FRET pair, SDM E309A | U1250 |
| FRET pair, Q491A | U1250 |

## 13/10/18

**Felix, Jolijn**

Inoculations were spun down, washed once with 1x PBS and resuspended in 1x PBS. 500 μg coerentazine was solubilized in ethanol to reach a final concentration of 7.5 mM. An aliquot was taken from this stock to make a 30x solution with 1x PBS. 480 μl bacteria were put in a 400 μl quartz cuvette and 20 μl 30x coerentazine solution was added. The solution was oxygenated using a pipette. No luminescence could be observed, indicating that the coelenterazine is not oxidized by luciferase. Fluorescence measurements of a wavelength that excites YFP, proved YFP to be present.

**Jolijn**

## 17/10/18

Samples that were transformed on 12/10/18, consisting of both the BRET pair and the tar receptors with altered amino acid sequence (to mimic methylation) were inoculated in LB.

**Jolijn**

## 17/10/18

The inoculated samples were transferred to a citrate phosphate buffer (0.15 M, pH 5). Coelenterazine was added to a final concentration of 7.5 µM. Measurements were performed, there was no measurable luminescence.

## 30/07/18

**Jolijn**

### Transformation of Tar part 1

The Tar, Tar GFP and Tar GFP His plasmids received on a filter paper from team Technion Israël 2016 were dissolved in 80 µL elution buffer. ~40 µL of plasmid dissolved in elution buffer was extracted. Competent DH5α cells were then transformed with the dissolved plasmids according to the iGEM protocol. Since the type of antibiotic resistance was unknown, Kanamycin, Ampicillin and Chloramphenicol were used as antibiotics.

## 31/07/18

**Jolijn**

### Transformation of Tar part 2

The transformation of Tar GFP His on chloramphenicol was the only successful transformation. Two colonies were picked and inoculated in 5 ml LB containing chloramphenicol.

## 01/08/18

**Jolijn**

### Transformation of Tar GFP His part 3

Tar GFP His DNA was isolated from the inoculation (31/07/18) according to the MiniPrep [protocol](https://2018.igem.org/Team%3AUtrecht/Protocols#PlasmidPurification). An aliquot of the left bacteria was plated in fresh agar plates.

### Transformation of Tar part 1

The DNA concentration of the Tar, Tar GFP and Tar GFP His samples were measured, concentrations are listed in the table below.

| Sample | Concentration |
| --- | --- |
| Tar | 10.4 µg/mL |
| Tar GFP | 9.1 µg/mL |
| Tar GFP His | 11.1 µg/mL |

The desired concentration of DNA for transformation is 100 ng in a 50 µL solution. Due to the low concentration of our samples, 5 µL of DNA sample was added to the 50 µL DH5α competent cells. Additionally, the Tar receptor (6/4j) and Tar receptor with promotor (6/15B) plasmids from the distribution kit were transformed as described in the iGEM transformation protocol.

## 02/08/18

**Felix, Jolijn**

### Transformation of Tar part 2

All transformations were successful, with a high colony density. One colony per sample was inoculated for 6 hours. After 6 hours the colonies were put in 5 ml LB + chloramphenicol and incubated overnight 37°C 200 rpm. Samples were as described below.

|  |  |
| --- | --- |
| Tar A | Ta |
| Tar B | Tb |
| Tar Kit A | Tka |
| Tar Kit B | Tkb |
| Tar GFP A | Tga |
| Tar GFP B | Tgb |
| Tar GFP His A (Israel) | Tgha |
| Tar GFP His B (Israel) | Tghb |
| Promotor Tar Kit A | Ptka |
| Promotor Tar Kit B | Ptkb |

## 03/08/18

**Felix, Jolijn**

### Transformation of Tar part 2

DNA was retrieved from the following samples using miniprep according to [protocol](https://2018.igem.org/Team%3AUtrecht/Protocols#PlasmidPurification).

| Receptor | ug/ml |
| --- | --- |
| T B | 65.8 |
| T A | 66.5 |
| Tk A | 60.7 |
| Tk B | 75.4 |
| Tg A | 73.3 |
| Tg B | 69.1 |
| Tgh B | 61.6 |
| TghA | 77.7 |
| Ptk A | 68.6 |
| Ptk B | 78.9 |

Site Directed Mutagenesis (SDM) was performed to mimic constant methylation of the Tar receptors. The experiment was carried out according to the [SDM protocol](https://2018.igem.org/Team%3AUtrecht/Protocols#SDM). Only B samples from the duplo were used for SDM. Following primers were used to specifically mutate Q491A and E309A.

|  |  |
| --- | --- |
| E491A\_F | GCATCGCTGGTGCAGGCGTCAGCTGCCGCCGCC |
| E491A\_R | GGCGGCGGCAGCTGACGCCTGCACCAGCGATGC |
| Q309A\_F | CTGCCGCCAGCATGGAGGCGCTCACCGCGACAGTGAAG |
| Q309A\_R | CTTCACTGTCGCGGTGAGCGCCTCCATGCTGGCGGCAG |

## 06/08/18

**Jolijn**

The prior made (03/08/18) SDM plasmids were transformed *in duplo* into DH5α cells according to the iGEM protocol.

## 07/08/18

**Felix**

Transformation was successful. The colony density was very high, which made it difficult to pick specific colonies for further incubation. Yet, colonies were picked and activated in 1 ml LB + chloramphenicol for 5 hours. After 5 hours no bacterial growth was observed.
Colonies were picked again and inoculated overnight in 5 ml LB + chloramphenicol. To ease colony picking, a colony of each sample was picked roughly and spread and diluted on a new agar plate + chloramphenicol. This plate was incubated overnight at 37°C.

## 08/08/18

**Jolijn, Felix**

The colonies picked on 07/08/18 were diluted successfully. The plasmid DNA of the inoculations (07/08/18) was purified according the [miniprep protocol](https://2018.igem.org/Team%3AUtrecht/Protocols#PlasmidPurification).

Following concentrations were observed.

| Receptor | Concentration ug/ml |
| --- | --- |
| Tb2 | 43.6 |
| Tb1 | 49.1 |
| Tgb2 | 52.7 |
| Tgb1 | 28.1 |
| Tkb2 | 46.33 |
| Tkb1 | 46.3 |
| Tgh2 | 15.3 |
| Tgh1 | 50.8 |
| Ptkb2 | 3 |
| Ptkb1 | 32.6 |

Tb2 and Tgb2 were sent for sequencing, although DNA concentration was not optimal.
DNA concentrations were not satisfactory, new colonies were picked and inoculated in 5 ml LB with chloramphenicol. A control was included to compare retrieved DNA concentrations.

## 09/08/18

**Jolijn, Felix**

DNA from inoculations was retrieved using miniprep according to the protocol. One modification was applied, the elution buffer was heated (60°C) before use and only 30 µl was applied on the spin column.

| Receptor | Concentration (µl/ml) |
| --- | --- |
| T1 | 102.9 |
| T2 | 106.2 |
| Tg1 | 85 |
| Tg2 | 110.2 |
| Tk1 | 33.9 |
| Tk2 | 68.8 |
| Ptk1 | 13.3 |
| Ptk2 | 71 |
| Tgh1 | 48.2 |
| Tgh2 | 49.1 |
| Control (Vector = PbsK) | 428.4 |

## 13/08/18

**Felix**

TG, TG2, T1,T2 were sent for sequencing.

## 14/08/18

**Jolijn**

Sequencing data revealed presence of GFP in all the sent sequences.

## 27/08/18

**Jolijn**

### Colony picking of E. coli

*E. coli* used for the interlab study were picked and inoculated for an hour.

### Genomic PCR

The obtained samples were used for colony PCR. Afterwards, the samples were loaded on a 0.8% agarose gel at 120 V for 20 minutes. Bands were isolated and purified, using the gel purification protocol. The concentrations were too low to continue (1.3µL and 1.9µL).

## 29/08/18

**Jolijn**

Receptor assay inoculations (custom receptor//Cu-Promoter::RFP sample 13 and 14 were used for genomic PCR. 1 mL of these cell cultures were used and spun down on 12000 x g for 1 minute. The supernatant was removed and resuspended in 100 µL milli-Q, after which custom 13 was heated at 85 °C’. The samples were diluted 0, 10 and 100 times and the resulting dilutions were used for 1-Taq PCR using the KOD polymerase [protocol](https://2018.igem.org/Team%3AUtrecht/Protocols#PCR).

Next, 50 µL loading buffer was added and all samples from custom 13 and 14 were loaded in lane 2 and lane 3 of a 0.8% agarose gel respectively. The bands were isolated and put together. The DNA concentration was 146.1 µg/mL.

## 30/08/18

**Jolijn**

### T4 PNK Phosphorylation and T4 ligation

The DNA fragments were phosphorylated according to the T4 PNK Phosphorylation protocol Using the following pipet scheme:

| Compound | Volume |
| --- | --- |
| DNA | 15 µL |
| 10x buffer | 2 µL |
| ATP 10 mM | 2 µL |
| T4 PNK | 1 µL |

Next, ligation was done in order to ligate the isolated Tar receptor DNA into the pBSK vector. This was done according to the following pipetting scheme:

| Compound | Volume |
| --- | --- |
| T4 ligase | 0.9 µL |
| Buffer | 1 µL |
| Vector | 1 µL |
| DNA | 7.60 µL |

After four hours of incubation, competent DH5α cells were transformed with the sample in duplo according to the iGEM transformation protocol.

## 11/09/18

**Jolijn, Lorenzo**

Transformation was successful, colonies were picked. Cells were incubated in 4 mL LB with 4 µL ampicillin.

## 30/09/18

**Lorenzo**

Tar receptor received from Groningen and transformed into DH5a according to the protocol.

## 03/10/18

**Lorenzo**

Tar receptor DNA was isolated using miniprep, DNA concentration was 356.3 ng/ul.
First site directed mutagenesis was performed (E491A) using phusion polymerase and listed HPLC purified primers.

|  |  |
| --- | --- |
| E491A\_F | GCATCGCTGGTGCAGGCGTCAGCTGCCGCCGCC |
| E491A\_R | GGCGGCGGCAGCTGACGCCTGCACCAGCGATGC |
| Q309A\_F | CTGCCGCCAGCATGGAGGCGCTCACCGCGACAGTGAAG |
| Q309A\_R | CTTCACTGTCGCGGTGAGCGCCTCCATGCTGGCGGCAG |

## 04/10/18

SDM1 (E491A) was digested with DpnI for 1.5 hours and heat inactivated for 30 min.
SDM2 (Q309A) was performed with SDM1 (E491A) as template. As the DNA concentration could not be retrieved, a titer of three different DNA concentrations was in three different DNA volumes:

* 0.3 ul
* 0.7 ul
* 1.1 ul

SDM1 (E491A) and SDM 2 (Q309A) were transformed in DH5a. During transformation, heat shock was performed inaccurately, therefore, the transformation was performed again.

1.1 (E491A) → SDM 1
2.1 (Q309A) → SDM A 0,3 ul
2,1 (Q309A) → SDM B 0,7 ul
2,1 (Q309A) → SDM C 1,1 ul

Second Transformation:

1.1 (E491A) → SDM 1 second time
2.1 (Q309A) → SDM 2.1 A second time 0.3 ul
2.1 (Q309A) → SDM 2.1 B second time 0,7 ul
2.1 (Q309A) → SDM 2.1 C second time 1.1 ul

## 05/10/18

**Felix**
Several transformations were successful.
Two colonies from the following transformations were inoculated
1.1 (E491A) first time
1.1 (E491A) second time
2.1 (Q309A) B 0,7 ul

## 05/10/18

**Felix, Lorenzo**

DNA from the following samples was purified according to the miniprep protocol.

DNA 6/15B (Groningen) (DNA 6/15B → SDM 1.3 (E491A))
SDM (E491A) 1.2 A (SDM 1.2 A → SDM 2.2 (Q309A) A)
SDM (E491A) 1.2 B (SDM 1.2 B → SDM 2.2 (Q309A) B)

Following samples were transformed.

Chloramphenicol + Ampicillin, U1250

* pVS88 + Tar SDM 1.2 (E491A) A
* pVS88 + Tar SDM 2.1 (Q309A) A
* pVS88 + Tar SDM 2.2 (Q309A) A
* pVS88 + Tar SDM 2.2 (Q309A) B

Chloramphenicol DH5α

* Tar SDM 2.2 A (Q309A) (Not digested with DpnI overnight)
* Tar SDM 2.2 B (Q309A) (Not digested with DpnI overnight)
* Tar SDM 2.1 A (Q309A)
* Tar SDM 2.1 B (Q309A)
* Tar SDM 2.1 C (Q309A)
* Tar SDM 1.3 (E491A)

## 07/10/18

**Jolijn**

Following sequences were transformed according to the iGEM protocol to DH5α

* SDM 2.1 (Q309A) C
* SDM 2.2 (Q309A) A after overnight DpnI
* SDM 2.2 (Q309A) B after overnight DpnI

Transformations were succesfull

 **Following sequences were inoculated**

* CheZ Rluc A (1)
* CheZ Rluc B (1)
* CheZ Rluc A (2)
* CheZ Rluc A (3)
* CheZ Rluc B (2)
* SDM 2.1 (Q309A) B
* SDM 1.3 (E491A) (1)
* SDM 1.3 (E491A) (2)

![](https://static.igem.org/mediawiki/2018/5/58/T--Utrecht--2018-MO7-1-Notebook.jpg)
![](https://static.igem.org/mediawiki/2018/b/b5/T--Utrecht--2018-MO7-2-Notebook.jpg)
![](https://static.igem.org/mediawiki/2018/a/a6/T--Utrecht--2018-MO7-3-Notebook.jpg)
![](https://static.igem.org/mediawiki/2018/a/a8/T--Utrecht--2018-MO7-4-Notebook.jpg)
![](https://static.igem.org/mediawiki/2018/7/7b/T--Utrecht--2018-MO7-5-Notebook.jpg)
![](https://static.igem.org/mediawiki/2018/f/fe/T--Utrecht--2018-MO7-6-Notebook.jpg)
![](https://static.igem.org/mediawiki/2018/1/1b/T--Utrecht--2018-MO7-7-Notebook.jpg)
![](https://static.igem.org/mediawiki/2018/6/6b/T--Utrecht--2018-MO7-8-Notebook.jpg)

## 08/10/18

**Felix**

DNA was acquired from samples according to the miniprep protocol with the following DNA concentrations.

| Sample | Concentration (ng/μl) |
| --- | --- |
| SDM 2.1 (Q309A) B | 171.1 |
| SDM 1.3 (E491A) (1) | 212.2 |
| SDM 1.3 (E491A) (2) | 110.3 |

**Inoculation**

Transformations: SDM 2.2 (Q309A) A after overnight DpnI, SDM 2.2 (Q309A) B after overnight DpnI, SDM 2.1 (Q309A) C (1.1 μl), were successful.

![](https://static.igem.org/mediawiki/2018/b/b8/T--Utrecht--2018-MO9-1-Notebook.jpg)
![](https://static.igem.org/mediawiki/2018/4/4e/T--Utrecht--2018-MO9-2-Notebook.jpg)

Two colonies were picked from the following samples:

- SDM 2.2 (Q309A) A after overnight DpnI
- SDM 2.2 (Q309A) B after overnight DpnI

One colony was picked from

* SDM 2.1 (Q309A) C (1.1 μl)

## 09/10/18

**Felix**

DNA from the following inoculations was purified

* 2x SDM 2.2 (Q309A) A after overnight DpnI
* 2x SDM 2.2 (Q309A) B after overnight DpnI
* 1x SDM 2.1 (Q309A) C (1.1 μl)

The following concentrations were measured and sent for sequencing.

| Sample | Concentration (ng/μ) | Sent for Seq. |
| --- | --- | --- |
| SDM 2.1 (Q309A)  C | 253.2 | VF: 1BA9ZAB682 |
|  |  | VR: 1BA9ZAB681 |
| SDM 2.2 (Q309A) A | 188 |  |
| SDM 2.2 (Q309A) A2 | 309 | VF: 1BA9ZAB678 |
|  |  | VR: 1BA9ZAB677 |
| SDM 2.2 (Q309A) B | 219.7 | VF: 1BA9ZAB680 |
|  |  | VR: 1BA9ZAB679 |
| SDM 2.2 (Q309A) B2 | 173.7 |  |

## 10/10/18

**Felix**

Analysis of the sequences revealed that none of the samples contained the desired DNA sequence with the specific mutation.

## 11/10/18

**Felix, Lorenzo**

Again SDM was performed (SDM Q491A as well as SDM E309A) according to the protocol using phusion polymerase with lower primer concentrations of vector DNA. SDM Q491A as well as SDM E309A were transformed in DH5α according to the protocol. The two SDM samples were also transformed in U1250 with the BRET pair (lab journal BRET).

## 11/10/18

**Felix**

SDM transformations were successful. 2 colonies from each SDM sample were picked and inoculated in 5 ml LB with chloramphenicol. SDM transformation in combination with the BRET pair were successful as well (lab journal BRET).

![](https://static.igem.org/mediawiki/2018/f/f1/T--Utrecht--2018-MO12-1-Notebook.jpg)
E309A
![](https://static.igem.org/mediawiki/2018/8/8e/T--Utrecht--2018-MO12-2-Notebook.jpg)
Q491A

[Project Design](https://2018.igem.org/Team%3AUtrecht/Design)

[Results](https://2018.igem.org/Team%3AUtrecht/Results)

[Protocols](https://2018.igem.org/Team%3AUtrecht/Protocols)



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# Team:Utrecht/Results

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Top

* [Introduction](#Introduction)
* [Receptor](#Aim1)
* [BRET](#Aim2)
* [Methylation](#Aim3)

![](https://static.igem.org/mediawiki/2018/b/bf/T--Utrecht--2018-HeaderResults.svg)

DeTaXion is a bacterial-based sensor for the detection of pharmaceutical contaminants in surface water. In its final form, DeTaXion will consist of a series of bacteria that can detect a wide variety of compounds. This will be achieved by engineering the chemotaxis pathway of *Escherichia coli* to emit a visible light signal in response to different pharmaceutical compounds. The project consists of three main aims:
Aim 1: Explore the possibility of using the ligand binding domains from different transmembrane receptors.
Aim 2: Development of a Bioluminescence Resonance Energy Transfer (BRET) readout for the activity of the chemotaxis pathway.
Aim 3: Adjusting the dynamic range of the chemotaxis system to enable detection of a broad range of contaminant concentrations.
Below we describe the results in each of these aims, including a description of the successful use of our submitted parts BBa\_K2736101 and BBa\_K2736105.

### Explore the possibility of using the ligand binding domains from different transmembrane receptors.

#### Approach

In its final form DeTaXion will use customized receptors that can detect different compounds. These receptors will be designed from a variety of existing transmembrane receptors (from the chemotaxis and other pathways), and further engineered with the help of prediction software such as Haddock, to provide the best possible binding affinity between receptor and ligand.

To provide a proof of concept within the scope of this project, we tested the possibility of swapping ligand binding domains.

![](https://static.igem.org/mediawiki/2018/5/5e/T--Utrecht--2018-Figure1-Results.png)

Figure 1: Schematic representation of the Tar receptor, CusS receptor, and our Hybrid receptor. Both Tar and CusR receptors contain a HAMP domain, involved in signal transduction to a kinase control domain. Based on the homology between the HAMP domains, we designed a hybrid receptor based on the extracellular domain of the Tar receptor and intracellular domains of the CusS receptor.

#### Results

We created a hybrid receptor consisting of the intracellular domains of the bacterial copper sensing protein CusS, fused to the ligand binding domain (LBD) of the Tar receptor (Figure 1). The Tar receptor is one of the main receptors of the chemotaxis pathway, and senses Aspartate (Bi et al., 2013). CusS is part of a two-component signaling pathway and senses copper and silver ions. In the presence of copper, CusS phosphorylates the response regulator CusR, which in turn interacts with a copper responsive promoter element, activating gene expression (Munson, Lam, Outten, & O’Halloran, 2000). We chose this configuration because it would allow us to test the possibility of generating hybrid receptors using a simple transcriptional readout, without having to be dependent on Aim 2 (generation of a functional BRET pair).

To test the functionality of our receptor, we generated a new BioBrick (BBa\_K2736105) consisting of a copper sensitive promoter followed by the coding sequence of the red fluorescent protein (RFP, from BBa\_K516032) (Figure 2). By measuring the intensity of RFP emission in a photospectrometer or by fluorescence microscopy, we can determine both whether the receptor functions, as well as to what degree it transduces a signal.

We transformed *E. coli* strain DH5α with plasmids encoding the RFP reporter, as well as our hybrid receptor. As a control, we transformed *E. coli* with the reporter alone. We grew both strains overnight in LB, which contains Aspartate and hence should result in RFP expression when our hybrid receptor is present. After overnight culture, we assessed the presence or absence of RFP by fluorescence microscopy. We observed bright RFP expression in bacteria transformed with both receptor and reporter encoding plasmids, but only very low levels of RFP in bacteria transformed with the reporter construct alone (Figure 3).

![](https://static.igem.org/mediawiki/2018/c/c3/T--Utrecht--2018-Figure2-Results.png)

Figure 2: Reporter construct consisting of RFP under control of the CopA copper-sensitive promoter (BioBrick BBa\_K2736105).

To further test the responsiveness of the Hybrid receptor to Aspartate, we attempted to grow our *E. coli* strain in a minimal medium lacking Aspartate (M9 + 0.4% glucose), which should not induce expression of RFP. However, we were unsuccessful at culturing bacteria in this medium. As an alternative, we grew bacteria overnight in LB (causing RFP expression), and then transferred them to M9 + 0.4% glucose for 8 hours to allow part of the RFP to degrade. We then split the bacteria over two tubes, and added 250 uM Aspartate to one of these samples. After 4 hours, we quantified the levels of RFP expression using a spectrophotometer. Even after 8 hours in M9, levels of RFP were still very high. Nevertheless, we observed slightly elevated levels of RFP in the bacterial culture incubated with Aspartate, compared to untreated control (Figure 4). While this indicates that our receptor is responsive to Aspartate, this result will need to be strengthened in future experiments.

![](https://static.igem.org/mediawiki/2018/b/b8/T--Utrecht--2018-Figure3-Results.png)

Figure 3: Hybrid Tar/CusS receptor induces expression of RFP. (A) Expression of RFP in bacteria expressing Hybrid receptor and carrying reporter construct. (B) Expression of RFP in bacteria carrying the RFP reporter alone. Scale bars are 10 um.

![](https://static.igem.org/mediawiki/2018/1/1d/T--Utrecht--2018-Figure4-Results.png)

Figure 4: Emission spectrum of bacteria expressing Hybrid receptor (BBa\_K2736109) and RFP reporter (BBa\_K2736105). Red line corresponds to bacteria treated with Aspartate, and blue line corresponds to untreated bacteria. Samples were illuminated at 585 nm. RFP peak emission is at 607 nm.

#### Conclusion

The expression of RFP in bacteria transformed with hybrid receptor and RFP reporter, but not in bacteria transformed with reporter alone, demonstrates the functionality of our copper-sensitive RFP reporter (submitted part BBa\_K2736105). Is also shows that its expression is dependent on the presence of the hybrid Tar/CusS receptor. Presumably the expression is due to the presence of Aspartate in LB medium. The slight increase in RFP expression upon addition of Aspartate after incubation in M9 medium (lacking Aspartate) for 8 hours further indicates that our receptor is indeed responsive to Aspartate. Together, these results demonstrate the feasibility of using alternative ligand binding domains to broaden the range of targets detectable by DeTaXion. Key future experiments include further investigation of the Aspartate responsiveness of the hybrid receptor, and the generation of receptors carrying ligand binding domains engineered to bind pharmaceutical compounds.

### Aim 2: Development of a Bioluminescence Resonance Energy Transfer (BRET) readout for the activity of the chemotaxis pathway.

#### Approach

To detect the activity of the chemotaxis pathway, we designed a Bioluminescence Resonance Energy Transfer (BRET)-based sensor, inspired by a previously made chemotaxis BRET-pair (Cui et al. 2014). We fused Renilla luciferase (RLuc) C-terminally to CheZ, and eYFP to CheY N-terminally. The final form of DeTaXion will give a quantifiable BRET signal, which will be measured as the change in the ratio of RLuc over eYFP luminescence (R/Y). In our experiments we defined a positive BRET signal as a change in R/Y of at least 0.5% because this has been shown to represent a detectable BRET signal (Cui et al., 2014; Shimizu, Delalez, Pichler, & Berg, 2006).

#### Results

![](https://static.igem.org/mediawiki/2018/7/70/T--Utrecht--2018-Figure5-Results.png)

Figure 5: Expression of eYFP::CheY. (A) *E. coli* DH5 transformed with eYFP::CheY and (BBa\_K2736101) and CheZ::RLuc (BBa\_K2736103). (B) *E. coli* UU1250 transformed with eYFP::CheY and (BBa\_K2736101) and CheZ::RLuc (BBa\_K2736103). Scale bars are 10 um.

The coding sequences of the BRET pair proteins were created by Gibson assembly of the parts BBa\_K569017 and BBa\_I15017, and BBa\_K629003 and BBa\_I159001 to create eYFP::CheY (part BBa\_K2736100), and CheZ::RLuc (part BBa\_K2736102) respectively. Subsequently, 3A assembly was used to add the promoter part BBa\_K608003 to these coding sequences (parts BBa\_K2736101 and BBa\_K2736103 respectively). The resulting vectors were transformed to either *E. coli* DH5α, which expresses wild type Tar, or *E. coli* strain UU1250, which lacks the Tar gene.

To confirm expression of eYFP::CheY we imaged bacteria by fluorescence microscopy. Expression of YFP was clearly detectable, confirming the presence of eYFP::CheY (Figure 5).

We next incubated the bacterial cells in PBS containing 7.5 µM coelenterazine (CAS# 55779-48-1 Nanolight #3031), the substrate for Renilla luciferase. However, we did not observe any emission of light, neither by immunofluorescence microscopy nor using a photospectrometer. We hypothesized that this result was due to the low permeability of coelenterazine through the cell wall. However, due to time constraints we were unable to further investigate this.

#### Discussion

Our results demonstrate the expression of eYFP::CheY (BBa\_K2736101). However, we were not able to measure BRET activity due to lack of bioluminescence from Renilla luciferase. We suspect that this is due to an inability of the luciferase substrate coelenterazine to penetrate into *E. coli*, as coelenterazine is known to have low penetration of bacterial cells. Solving this issue is the key next experiment to be performed, followed by detection of BRET signal in the presence and absence of the ligand Aspartate.

### Aim 3: Adjusting the dynamic range of the chemotaxis system to enable detection of a broad range of contaminant concentrations.

#### Approach

To facilitate the measurement of multiple ranges of concentrations, we adjusted the sensitivity of the receptor. This approach is based on the methylation state of the Tar receptor, which influences its sensitivity to ligands. In the unmethylated state, the receptor efficiently transduces the ligand binding signal to the downstream pathway ([see project design](https://2018.igem.org/Team%3AUtrecht/Design)). Upon receptor methylation, the conformational change required to transduce the signal, is energetically less favorable. Therefore, when the same amount of ligand is present, the level of signal transduction is lower than in the unmethylated receptor state. The Tar receptor has four acidic residues that can be methylated (Q295, E302, Q309, E491). The methylation of glutamine (Q) or glutamic acid (E) residues can be mimicked by mutating these residues to alanine (A) (Krembel et al. 2015).

#### Results and discussion

Our [computational modeling](https://2018.igem.org/Team%3AUtrecht/Model) indicated that the mutation of the methylation sites should indeed result in altered sensitivity of the receptor. We therefore chose to generate three different methylation state Tar variants:

1. The wild type state, the unmethylated Tar receptor (QEQE, most sensitive);
2. A Tar receptor with one mimicked methylation site (QEAE, average sensitivity);
3. A Tar receptor with two mimicked methylation sites (QEAA, least sensitive).

We received the wild-type Tar receptor from a [collaboration with iGEM team Groningen](https://2018.igem.org/Team%3AUtrecht/Collaborations#Tar). We next used site-directed mutagenesis to introduce the Q309A and E491A mutations, resulting in BioBrick parts BBa\_K2736108 and BBa\_K2736106 respectively. Sequencing confirmed the successful generation of these components. However, because the BRET assay was not yet functional, we were unable to test the relative activity of these Tar receptors in the time frame of the competition.

#### References

Bi, S., Yu, D., Si, G., Luo, C., Li, T., Ouyang, Q., … Lai, L. (2013). Discovery of novel chemoeffectors and rational design of *Escherichia coli* chemoreceptor specificity. Proceedings of the National Academy of Sciences. <https://doi.org/10.1073/pnas.1306811110>
Cui, B., Wang, Y., Song, Y., Wang, T., Li, C., Wei, Y., … Shen, X. (2014). Bioluminescence resonance energy transfer system for measuring dynamic protein-protein interactions in bacteria. mBio. <https://doi.org/10.1128/mBio.01050-14>
Krembel A, Colin R, Sourjik V (2015) Importance of Multiple Methylation Sites in *Escherichia coli* Chemotaxis. PLoS ONE 10(12): e0145582.
Munson, G. P., Lam, D. L., Outten, F. W., & O’Halloran, T. V. (2000). Identification of a copper-responsive two-component system on the chromosome of *Escherichia coli* K-12. Journal of Bacteriology. <https://doi.org/10.1128/JB.182.20.5864-5871.2000>
-Shimizu, T. S., Delalez, N., Pichler, K., & Berg, H. C. (2006). Monitoring bacterial chemotaxis by using bioluminescence resonance energy transfer: Absence of feedback from the flagellar motors. Proceedings of the National Academy of Sciences. <https://doi.org/10.1073/pnas.0510958103>

[Biobricks](https://2018.igem.org/Team%3AUtrecht/Parts)

[Project Design](https://2018.igem.org/Team%3AUtrecht/Design)

[Attributions](https://2018.igem.org/Team%3AUtrecht/Attributions)