Results

Overview

Construction of PCM(Population control module)

Cloning of pSB1C3

Aim: To procure higher quantities of pSB1C3 backbone that will be subsequently used for the NEBuilder® HiFi DNA assembly.

Experiment: We have used self-chemically prepared DH5-alpha chemically procured competent cells for cloning the revived part C-11 (kit plate 1, DistributionKit 2024) pSB1C3 via chemical transformation, did a plasmid extraction to get a better yield.

Result: We have successfully cloned the pSB1C3 vector through transformation and then done a plasmid extraction.

The concentration of the vector: 256.6 ng/ul (Nanodrop concentration)

PCR Amplification of the Gene Fragments

Aim: To PCR amplify the three fragments (from TwistBioScience) (code named by us as insert-1, insert-2, insert-3) so then we can perform the NEBuilder® HiFi DNA assembly of them with the pSB1C3 vector.

Experiments:

1. We did a gradient PCR to determine the annealing temperature for each fragment with the respective forward and reverse primers.

2. We proceeded with the optimal annealing temperature of the respective fragments to PCR amplify them.

3. For insert-2, we observed non-specific binding, which we managed to remove by running the gel 30 minutes more.

Result: 62.5 degrees Celsius was the optimal temperature for insert-1, insert-2, and insert-3.

Fig- the Gradient of the insert-1 and insert-2 Fig-The insert – 3 gradient PCR gel run and the desired bright band is on the 62 degree Celsius Fig- the amplified insert -3 gel run Fig- insert 1 and insert 2 circuit PCR amplified gel run Fig- the gel extraction of the PCR amplified plasmid Concentration of extracted plasmid= 56ng/u Fig-gradient PCR of the plasmid backbone Fig- Digestion of the plasmid (assembled) and the PCR run of the PCM (Main Construct) construct

To check the induction of the circuit we have performed an IPTG induction experiment which is further analysed in the quantitative assay.

Snap Gene file for the Entire Assembly – length 5908 bp

Fig- The Whole HiFi Assembled Circuit
By optimizing the previously mentioned protocol, we achieved successful cloning, which was further verified through single digestion at BamHI and colony PCR. These validation steps confirmed that the cloning was successful.

Characterisation of PCM Circuit

Cloning of the Circuit

Extraction of the GFP Plasmid

Aim – To procure higher quantities of sfGFP (superfolded GFP) that will be subsequently used for the NEBuilder® HiFi DNA assembly.

Experiment – We have used self-prepared DH5 alpha chemically procured competent cells for cloning the sfGFP via chemical transformation. Moreover, we did a plasmid extraction to get a better yield.

Result – We have successfully cloned the sfGFP sequence through transformation, followed by plasmid extraction. The concentration of the plasmid extracted sfGFP was 429 ng/ul.

Amplification of the GFP

Aim – To amplify the sfGFP sequence through the Polymerase Chain Reaction (PCR) for a future HiFi assembly.

Experiment – We have done a gradient PCR to determine the annealing temperature for sfGFP with the respective forward and reverse primers. We then proceeded with the optimal annealing temperature of the respective fragments to PCR amplify them. The optimal annealing temperature was 57 degree Celsius. The gradient PCRs for the sfGFP characterization of the tetR-AiiA sequence and sequence for Antisense DNA have been carried out with the following temperatures:– 52, 54, 56, 58, 60 degree Celsius.

Results – The desired band is observed at a length of around 757 bp. To get a better result in the gel electrophoresis experiment, we have used TAE buffer, which is not practically suitable for a long run. The gel was then run by reducing the voltage and increasing the time.

Linearisation for addition of GFP to the downstream of pLUX of AiiA for its characterisation (tetR)

Aim - To linearise the tetR-AiiA sequence characterisation part through PCR so as to obtain the backbone for insertion of sfGFP through NEBuilder® HiFi DNA assembly reaction.

Experiment - We have done a gradient PCR to determine the annealing temperature with the respective forward and reverse primers. We have then proceeded with the optimal annealing temperature of the respective fragments to PCR amplify them. The optimal annealing temperature is 57 degree Celsius. The gradient temperatures are set at: 50, 53, 56, 58, 60, 62 degree Celsius.

Result - We have achieved the desired length on gel imaging. We have obtained a successful circuit assembly through HiFi assembly of this vector.

Linearisation for addition of GFP in the place of Antisense for its characterisation (Antisense)

Aim - To linearise the Antisense characterisation part through PCR to insert sfGFP in the main circuit through NEBuilder® HiFi DNA assembly reaction.

Experiment - We proceeded with a gradient PCR to determine the annealing temperature for the linearised plasmid (up to the sequence of Antisense DNA) with the respective forward and reverse primers. We have then proceeded with the optimal annealing temperature of the respective fragments to amplify them with PCR reaction. The set gradient temperatures are 50, 53, 58, 60, and 62 degrees Celsius. The optimal annealing temperature is 58 degrees Celsius.

Result - We have achieved the desired length on gel imaging. We have obtained a successful circuit assembly through HiFi assembly of this vector.

Amplification of GFP in the pLUX of AiiA for its characterisation (tetR) and GFP in the place of Antisense sequence for its characterisation (Antisense)

Aim - To amplify the Antisense and tetR sfGFP characterisation part through PCR to get a better yield in the HiFi.

Experiment - We have proceeded with the optimal annealing temperature of the respective fragments (inferred from previous gradient PCRs) to PCR amplify them and then performed gel extraction in 0.8-1.5% agarose gel media. For gel extraction of length around 4-5 kb, we ran the gel on the TBE buffer and extracted the gel with 1.5% weight of isopropanol.

Result - We have observed that the gel bands have come on particular desired lengths. Being cautious while performing gel extraction brings us a step closer to a successful HiFi assembly.

Insert	Purity (ng/ul)
Gfp-tetR	38.8
Amp-tetR	35.4
GFP-anti	85.9
Amp-anti	32.8

HiFi assembly of the whole circuit

Aim - To assemble and characterize the full circuit besides checking whether it produces our protein of interest or not.

Experiment -

For Antisense sequence set up reaction on ice:

Component	Volume	Amount
Anti-amp (vector)	6.7 ul	0.067 pmoles
Sf-GFP	0.7 ul	0.133 pmoles
Deionised water	2.6 ul
NEBuilder HiFi DNA assembly Master Mix	10.0 ul

Total volume - 20 ul

For the tetR set up reaction on ice:

Component	Volume	Amount
tetR-amp (vector)	4.3 ul	0.067 pmoles
Sf-GFP	1.6 ul	0.133 pmoles
Deionised water	4.1 ul
NEBuilder HiFi DNA assembly Master Mix	10.0 ul

Total volume - 20 ul

The reaction mixtures will be incubated around 50 degrees Celsius for 15 minutes in the PCR machine.

Result - Colony PCR and quantitative assay analysis give positive results on production of the protein of interest.

The snap gene files of the above characterisation –

Name		Snap Gene file Length
GFP after pLUX of AiiA to characterise the circuit		4475 bp
GFP instead of antisense to characterise the circuit		6097 bp
sfGFP		717 bp
Amplified GFP		757 bp

Antisense

Experiments held to determine the best possible length of the cell cycle arrest (CCA) sequence (aka the Antisense)

Length of CCA Gene	Optimal Annealing Temperature (°C)
150 bp	60
277 bp	60
393 bp	66

Aim - to amplify the CCA gene sequence of different lengths from the 400 bp CCA as a part of the PCM construct through the PCR for the NEBuilder® HiFi DNA assembly into M20 pSB1C3SA backbone.

Experiment - A Gradient PCR (on a common gradient of annealing temperatures, across 54, 56, 58, 60, 62 deg C) was done to determine the annealing temperature for CCA gene for different length with the respective forward (FP common) and reverse primers (variable). Then we have proceeded with the optimal annealing temperatures of the respective fragments to PCR amplify them.

Result: The optimal annealing temperature is found to be 60, 60, 66 deg C for the 150 bp, 277 bp, 393 bp, respectively.

Fig: Gradient PCR of CCA of length 150 bp. Similarly done for the other two cases. (Ladder= NEB 1 kb+)

Amplification Of The Respective Different Lengths Of Antisenses/CCA

Aim - to amplify the Antisense three different lengths part through PCR to get a better yield in the HiFi result during NEBuilder® HiFi DNA assembly reaction.

Experiment - The amp. PCR protocol was troubleshot for small parts. The reaction was made to simulate ‘Hot Start’ – i.e., the reaction mixture was kept on ice. The PCR machine was preheated to 98 deg C and then paused. The reaction (100 µL, divided into two 50 µL tubes for a better reaction) tubes were then suddenly put into the PCR machine.

Result - The three lengths of the CCA were respectively run on suitable gels, and excellent bands at desired lengths were obtained, which were excised and immediately extracted with excellent yield (concentration) of DNA.

Fig- Amplification PCR Pdt. of CCA of 150 bp (Ladder= NEB 1 kb+) Fig: Amplification PCR Pdt. of CCA of 393 bp (Ladder= NEB 1 kb+) Fig: Amp. PCR Pdt. of CCA of length 277 bp (Ladder= NEB 1 kb+)

Linearization of Vector for CCA of Variable Lengths

Aim: To run gradient PCR for the linearization of the common backbone vector for each of our antisenses (Kit part M20, Kit Plate 1, Kit 2024).

Theory: The primers were based on the amplification of a major part, including the pTac promoter, lacO operator, and a terminator. The pTac was default suppressed due to lacI in the environment but can be switched on by IPTG. The idea was to exploit this kit part into our attempt at switching OFF - ON - OFF systems of our antisenses when we would assemble our antisenses (of various lengths) into this backbone.

Result: The best T_a for the M20 Backbone was found to be 63 deg C.

Aim: To linearize the M20 Backbone based on the T_a obtained earlier.

Experiment: A 100 µL reaction mixture, as written earlier, was set up. It was divided into two 50 µL reaction tubes for better PCR (a recent learning) and then loaded onto a single, big well for a total of 100 µL.

Result: Visualized on a low percent gel.

Fig: Linearization PCR Pdt. Of M20 Backbone (Ladder= NEB 1 kb+)

NEB HiFi Assemblies

HiFi 1: (M20+150bp)

Vector Length: 2,311 bp

Vector Source: M20, Kit Plate 1, Distribution Kit 2024

Insert	Length (bp)	Nanodrop Conc. Of Gel Extract (ng/uL)	HiFi Assembly used in
CCA of 150 bp	150+~40 overhang	62.25 (dil. 2X)	HiFi 1
CCA of 277 bp	277+~40 overhang	17.9	HiFi 2
CCA of 393 bp	393+~40 overhang	79.8	HiFi 3

The following reaction mixtures were set up:

HiFi 1

• M20 Vector: 1 µL
• Insert 150 bp: 0.3 µL
• NEB HiFi Builder MasterMix: 6 µL
• Deionized H₂O: 12.7 µL
• Total: 20 µL

HiFi 2

• M20 Vector: 1.26 µL (0.05 pmolar)
• Insert 277 bp: 0.6 µL (1 pmolar)
• NEB HiFi Builder MasterMix: 6 µL
• Deionized H₂O: 12.2 µL
• Total: 20 µL

HiFi 3

• M20 Vector: 2.8 µL
• Insert 393 bp: 0.24 µL
• NEB HiFi Builder MasterMix: 6 µL
• Deionized H₂O: 12.6 µL
• Total: 20 µL

The backbone, the fragment (either of antisense 150 bp, 277 bp, 393 bp), MasterMix, and NFW were incubated in the PCR machine at 50 deg C for 30 min.

Quantitative Assay

Aim – to observe induction of the IPTG inducible promoter pTac and its transcription unit in the PCM (Main Circuit) through a growth curve measurement of the PCM-transformed bacterial culture.

Experiment – a spectrophotometric growth curve analysis, with IPTG induction. Three identical secondary cultures- one control, one with IPTG induction at 0th time point, and another with IPTG induction after 2 hours of setting the secondary culture, were given, for the E. coli strain BL21 cells transformed with PCM/Main Construct Hifi Assembled plasmid.

Result- there is a minute deserved oscillation in the circuit is observed.

Aim- to observe how different concentration of inducer IPTG affected the growth and production of the CCA (Cell Cycle Arrest) sequence (aka the Antisense)

Exp.- in a 24 well microplate with a different concentrations of IPTG – 0.1 mM , 0.5 mM and 5 mM- we have done the assay for the both control (revived part M20, kit plate 1, 2024 Kit ) and PCM construct (aka the Main Construct).

Result- the induction in the circuit is not that prominent but a minute decline in the stationary phase population was observed wrt control.

Aim- to observe how different concentration of inducer IPTG , AHL and aTc (anhydrous Tetracycline) affected the growth and production of antisense (CCA)

Experiment- in a 96 well microplate with a different concentration of IPTG – 0.1 mM , 0.5 mM and 5 mM we have done the assay for the both control M20 ( well plate 1 ) and PCM construct now we have given diff concentration of AHL and same concentration- aTc (anhydrous tetracycline)

Result- Fig - the aTc induced circuit shows an observable fluctuation. (HiFi denotes the Main Construct or the PCM) Fig- the without atc induced circuit show an observable fluctuation.

Aim- characterisation of the circuit through fluorophore sfGFP through the fluorescence analysis

Experiment- in a well microplate with a different concentration of IPTG – 0.1 mM , 0.5 mM and 5 mM we have done the assay for the both control M20 ( well plate 1 ) and PCM construct.

We replaced tetR_AiiA-dtag under pTac promoter, in our construct- the PCM, with sfGFP, and code-named the construct as tetR. It showed the following growth curve with IPTG induction for the 1 mM case. Fig- the induced tetR or the circuit is successfully characterised in presence of IPTG Fig- the induced tetR is successfully characterised in presence of IPTG. Fig- the induced ‘anti’ circuit (sfGFP replacing CCA sequence downstream of second pLux promoter) is successfully characterised in presence of IPTG.

Oscillatory growth curve was achieved in different length analyses, quite in line with our expectations.

Analysis of the Resultant data We eventually fitted our wetlab data obtained from 0.1nM IPTG+AHL induction with the math model for quorum sensing and came up with the following fitted plot. For an insight into the fitted parameters, visit DryLab

We did not observe significant changes in fluorescence during our assay, indicating that the circuit did not function as expected and the components were not properly fine-tuned. This opens the opportunity for circuit modifications, where we plan to introduce degradation tags to various components to better fine-tune parameters based on a revised model. Initial predictions, based on our circuit's original parameters, forecasted oscillations, but experiments revealed no oscillations or notable phenotypic changes. This failure could be due to signals being too weak for detection by our instruments. However, we did demonstrate that induction of the cca gene caused an arrest, which was released after a specific time interval. The mechanism behind this release remains unclear, but we have designed experiments to explore it further.

Fungal Assay

Aim- to check the growth of the fungi (Chaetomium thermophilum ) with respect to different concentration of the commercial linalool (monoterpenoid).

Experiment-to check the growth we need to prepare a suitable media for fungus

Mycelium basically propagates on the CCM medium.To prepare this we need to follow the following composition:

Procedure:

1. We need to grow the fungus overnight in a culture and through the pipette pluck one single colony in the centre of the plate.

2. After that, marking the same distance with the scale, we need to put the single colony in the plates varying the concentration of the Linalool on the plates.

3. The plates need to be incubated under 45 degrees centigrade at 250 rpm.

4. After 24 hrs, we have to check the growth and measure the lengths of the radius to get a rough idea about the growth of mycelium.

Result:

With the varying concentration of Linalool, we have found that at a certain level of concentration, fungal growth has been stopped. The below observations are after 24 hrs.

Fig- after treatment with 0 linalool mycelia growth diameter is 55 mm Fig- after treatment with 56 ug/mL the mycelia growth diameter is 30 mm Fig- after treatment with 150 ug/mL Linalool the mycelia growth diameter is 25 mm Fig-After treatment with 256 ug/ml Linalool no growth observed