21/08/18

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

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

23/08/18

Miniprep was performed according to the protocol.
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 80C 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 80C 20 min.

DNA was transformed in DH5a according to the iGEM protocol.

24/08/18

Transformation was successful 8 colonies were picked to perform colony PCR according to the protocol. (Cu-promotor::RFP 100%, Cu-promotor::RFP 500%, Custom receptor 100%, Custom Receptor 500%).

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

27/08/18

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

28/08/18

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.

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

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

The DNA of the (28/08/18) samples was purified according to the miniprep protocol.

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)

30/08/18

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.

05/09/18

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

The Cu::RFP DNA was purified according to the miniprep protocol with 5 ml bacteria instead of 1.5 ml and eluted in 20 µl elution buffer. DNA was sent for sequencing.

10/09/18

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

11/09/18

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

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

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

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

22/09/18

Since our previous attempts didn’t yield any results we decided to place the copper promoter gBlock was cut 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

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

The reaction mix was inactivated by heating it to 80 degrees 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

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

26/09/18

The DNA of the inoculated cultures was minipreped according to the miniprep protocol. 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.

29/09/18

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 Dh5a.

30/09/18

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.

01/10/18

100 µl of the overnight cultures from 09/30/18 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 degrees. 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.

02/10/18

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

03/10/18

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

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

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

Cu-promoter :: RFP Normarski

Cu-promoter :: RFP Nomarski Fluorescence

Cu-promoter :: RFP // Custom receptor

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

14/10/18

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 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 (10/13/18) 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.

30/07/18

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

* 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

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

01/08/18

Isolated the plasmids containing the biobricks from the inoculated colonies from 31/07/18 according to the plasmid purification protocol. (for concentrations see table below)

Performed a PCR, as described in the PCR protocol, 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.

02/08/18

Performed a gel electrophoresis on the PCR product from 01/08/18 on a 1.5% agarose gel according to the gel electrophoresis protocol. The resulting bands were isolated according to the gel purification protocol.

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.

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

03/08/18

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.

06/08/18

The CheZ and RLuc, and LuxA and LuxB fragments were fused together using Gibson assembly as described in the gibson protocol. 9 µl of each Gibson product was transformed to 100 µl of DH5ɑ, since this increases the amount of successful transformations.

07/08/18

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. These bacteria were inoculated overnight at 37 °C in a rotary shaker.

08/08/18

DNA of inoculations (07/08/18) was purified according the miniprep protocol. The acquired DNA was sent for sequencing.

09/08/18

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

10/08/18

PCR product from 09/08/18 was ran on 0.8% gel and isolated the DNA using the Gel purification protocol

15/08/18

eYFP::CheY was assembled according to the Gibson cloning protocol. The cloning product was transformed into DH5α according to the iGEM transformation protocol.

16/08/18

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. Positive samples were selected and inoculated overnight.

17/08/18

20/08/18

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

27/08/18

were sent for sequencing with m13 forward and reverse primer

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

28/08/18

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.

The fragments were used to perform a Gibson assembly of eYFP::CheY, and 9 µl of the product was transformed to 100 µl DH5a.

29/08/18

We checked the plates from 28/09/18 but none of the transformations were successful.

03/09/18

We performed the gibson reaction of eYFP::CheY again. The gibson product was transformed directly to DH5a. 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

Since our the 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 DH5a.

12/09/18

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

12/09/18

DNA from inoculations was isolated according to the miniprep protocol. 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 DH5a.

14/09/18

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

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

06/10/18

Oligos containing the promoter CheZ::RLuc part sequence were annealed according to the annealing protocol

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.

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.

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

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.

08/10/18

DNA was purified from the following samples using the miniprep protocol and sent for sequencing.

The Gibson cloning was transformed in DH5α according to the protocol.

09/10/18

Transformation was successful

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

10/10/18

DNA from the two inoculation of 1/5A::CheZ::Rluc after gibson cloning was retrieved with miniprep according to the protocol in the following concentrations.

11/10/18

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:

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

12/10/18

SDM transformations (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 10/11/18.

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).

The following samples were transformed

13/10/18

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.