Basal Expression of LasI (Module 1)

Cloning the construct and Confirmation

We intend to find the Basal expression of LasI. We cloned our genetic circuit into pJUMP, our destination plasmid, with the golden gate assembly.

The size of the cloned plasmid is 3499bp.

We did colony PCR to confirm whether bacteria had taken up the cloned plasmid.

We designed colony PCR primers in a way where we had two amplicon sizes within the plasmid.

The expected sizes of amplicons are �-

Plasmid Forward and Insert Reverse 528bp

Insert Forward and Plasmid Reverse 824bp

Plasmid Isolation and Restriction Digestion

We performed restriction digestion with SpeI, EcoRV, and EcoRI enzymes to further confirm the colony.

The Expected sizes are 2469, 855, 175.

BL-21 Transformation and Confocal microscopy

We also checked under an epifluorescence microscope to confirm the positive colonies.

We used a confocal microscope to check the basal expression.

We inferred that there is some basal expression of LasI.

Expression of SNAT and COMT (Module 6)

Cloning the construct and Confirmation

We planned to check the expression of SNAT and COMT by tagging them with TagBFP and mCherry respectively. We cloned our genetic circuit into pJUMP by Golden Gate assembly using the BsaI restriction enzyme.

• The size of the cloned plasmid is 5833bp.

• We performed colony PCR to check the positive colony.

The expected sizes of amplicons are

Plasmid forward and insert reverse 1248bp

Insert forward and Plasmid Reverse 1063bp

Plasmid Isolation and Restriction Digestion

We performed restriction digestion with HindIII and EcoRV single-cutter enzymes to further confirm the colony, both enzyme sites were present in the insert at different locations.

The expected band size is 5833bp.

BL-21 Transformation and Confocal Microscopy

The plasmid was transformed into BL-21 and colonies were observed. The culture was induced by the addition of IPTG and the expression of SNAT and COMT with IPTG induction was checked by confocal microscope.

We concluded that SNAT and COMT can be expressed under an inducible promoter like pLasI.

The Serotonin Induced regulatory system (Module 4)

Cloning the construct and Confirmation

We opted for Golden Gate assembly to assemble the parts in our plasmid. We used the BsaI restriction enzyme to produce overhangs and run the reaction in a Thermocycler to complete the Golden Gate assembly using the protocol on our Protocols page. We have ordered colony PCR primers to confirm our cloning through colony PCR and Gel electrophoresis. The colony PCR results are as below for this module:

• Gel electrophoresis - we got 1:5 5th colony positive result with successful clones.

• Correct bands were observed in the 9th lane of the 1st gel (1.6 Kb) and the 7th lane of the 2nd Gel(1.2 kb).

• 7th lane of 2nd Gel- 1:5 (Vector: Insert ratio), 5th Colony, Part a 9th lane of 1st gel- 1:5 (Vector: Insert ratio), 5th Colony, Part b

• Colony 5 (1:5)- Positive

Plasmid Isolation and Restriction Digestion

Restriction Digestion of the isolated plasmid from DH5-$\alpha$ with EcoRI, NdeI, and SpeI.

Gel electrophoresis results are as follows:

BL-21 Transformation and Confocal Microscopy

The confirmed plasmid with the desired construct was transformed successfully into E. coli BL21 cells to check the expression and functionality of the regulatory system components. Confocal Microscopy was performed to confirm the fluorescence reporter mCherry present in this module which shows its expression after the Serotonin-LasR complex activates the transcription of downstream genes of the LasI promoter. Expected Results: Either Basal expression or no expression should be observed in the module when no inducer molecule - Serotonin is added. An increase in the intensity of mCherry should be observed when serotonin is added to the culture. We inoculated the primary culture of BL-21 cells which had the confirmed construct and after the OD600 nm reached 0.4 i.e., the exponential growth phase of the cells, inducer molecule - serotonin of 80 $\mu$M concentration (physiological levels of serotonin in IBS-D patients) and 100 $\mu$L volume of this solution is added into 10mL secondary culture. The confocal microscopy was performed at 0min, 40min, and 120mins and observed less significant change in the intensity after 40mins. All the parameters like exposure, emission, and absorption wavelengths were kept constant during the observations. The pictures below depict the intensities observed.

Inference - 1

The above result suggests that the regulatory system induced by Serotonin works well. In the presence of serotonin, as it is clearly visible from the Confocal Microscopy results, the presence of mCherry protein is seen after the addition of serotonin.

Inference - 2

The SERT analog CUW_0748 acts slower than the desired efficiency. As there is no significant increase in the intensities at 40 mins and at 120 mins, we could say that CUW_0748 should be expressed constitutively.

Bacterial Growth in the Presence of Serotonin.(Module 7)

Serotonin has an impact on the growth of bacteria in the gut. Literature reveals that in the presence of high levels of serotonin, it is difficult for bacteria to grow properly. To test this we cloned the whole 7th module and transformed it into BL-21 after partial confirmation of the presence of construct through restriction digestion. Colonies were observed in the Kanamycin selective BL-21 plate which leads to 2 inferences:

1. The presence of a cassette in the plasmid due to the presence of colonies but no colony PCR confirmation was obtained and hence we infer there might be a problem with colony PCR primers.

2. Partially correct results in the restriction digestion also infer the presence of cassette.

However, we continued with the colonies we had and checked the bacterial growth curve under the kanamycin selective marker and serotonin stress marker. The graph below shows that at 80 $\mu$M concentration of serotonin, the cells� growth had no significant impact.