<!DOCTYPE html> Engineering

Engineering Success

Due to the second wave of the pandemic in India, our wet lab work was considerably delayed, effectively leaving us with a mere month and a half in the lab. We utilised our time working remotely to devise a thorough plan of experiments, incorporating a Design-Build-Test cycle:

Choosing Optimal promoters

Cycle 1
Cycle 2

Cycle 1

Design	Design constructs via overlap PCR of various native promoters - Pcpc560, PcpcB-m6, PpsbA2, PpsbA3 - upstream of fluorescent reporters. Compare their promoter strengths to that of strong constitutive promoter J23119 and the IPTG-inducible lacUV5.
Build	Incorporate constructs via homology directed repair (HDR) into Neutral Site I of the genome using a CRISPR/Cas12a system.
Test	Vary light intensity and concentration of carbon dioxide and study fluorescence to characterize promoter activity (strength and response)
Learn	Select promoters with high activity and easily tunable response.

Cycle 2

Design	Design constructs with strong/tunable promoters from previous round, with cscB, sps and spp
Build	cscB is incorporated into Neutral Site I, and sps and spp into Neutral Site III, via HDR using a CRISPR/Cas12a system
Test	Collect supernatant samples over time to estimate sucrose production; use Dr Pakrasi lab’s strains as a positive control.
Learn	Choose promoters that maximize sucrose yields in the particular carbon dioxide and light conditions we choose to use in our future implementation.

We were unfortunately unable to carry out most of these experiments in the little time remaining to us. We instead acquired sucrose-secreting strains of S. elongatus UTEX 2973 from the Pakrasi lab, to measure their sucrose production rates. We have also acquired everything we need for future experiments according to our original plan.

gDNA extraction and PCR

Cycle 1

Design	To extract genomic DNA (gDNA) from E. coli KJK01 and verify the presence of hbd-crt genes by PCR
Build	Genomic DNA was extracted through the phenol-chloroform method, and PCR was performed to amplify the hbd-crt genes. The PCR product was then run on an agarose gel.
Test	The gel was imaged and no bands were observed.
Learn	There may have been a possible inhibition of PCR due to the excess concentration of dNTPs and gDNA in the PCR mix. This could be potentially fixed by reducing the concentrations in the subsequent attempt.

Cycle 2

Design	PCR with reduced concentrations of dNTPs and gDNA and quality check for gDNA
Build	The PCR product and gDNA were run on an agarose gel.
Test	The gel was imaged and multiple bands were observed in the well corresponding to the gDNA and no bands corresponding to the PCR product were visible.
Learn	Suspected nuclease contamination in gDNA sample

Cycle 3

Design	Modify the protocol such that Proteinase K is added first, followed by RNase A, after Proteinase K deactivation. Instead of Elution Buffer, perform the final gDNA resuspension in autoclaved ddH2O (MilliQ water).
Build	The gDNA was extracted with the modified protocol and run on an agarose gel.
Test	A single large band appeared above the highest band of the DNA ladder and large bright bands were present at the bottom of the ladder.
Learn	Good quality gDNA is present, and the large bright band may be due to RNA contamination.

Cycle 4

Design	PCR verification of hbd-crt from new gDNA extract
Build	The above was executed along with a negative control that was devoid of the gDNA template PCR products were run on an agarose gel
Test	No bands corresponding to the genes of interest appeared
Learn	RNA contamination may have led to an overestimation of the gDNA concentration when measured by NanoDrop, therefore the PCR should be redone with undiluted template gDNA. DMSO should also be added to the PCR mix to inhibit renaturation of the denatured DNA.

Cycle 5

Design	Gradient PCR with varying DMSO concentrations at various annealing temperatures
Build	The above was executed along with a negative control devoid of the gDNA template
Test	Band corresponding to genes of interest at 750 bp appeared
Learn	hbd-crt was amplified during PCR, its presence on the genome of E. coli KJK01 is verified

E. coli in CoBG11

Cycle 1

Design	Inoculate KJK01 and pCSCX-KJK01 cultures in BG-11 + sucrose to check for growth, sucrose consumption and butanol production. Sucrose concentration used is 250 mg/L, higher than maximum sucrose concentration secreted by cyanobacteria in Zhang et al, 2020¹.
Build	Inoculate 3 cultures • KJK01 (without antibiotics) • pCSCX-KJK01 (uninduced + antibiotics) • pCSCX-KJK01 (induced + antibiotics) Take hourly OD readings, store supernatants at -20°C and submit for sucrose assay and NMR analysis.
Test	KJK01 showed growth, while both pCSCX-KJK01 strains did not. Maximum sucrose was consumed by KJK01.
Learn	Suspected contamination in the KJK01 culture. Repeat experiment carefully to minimize all possibilities of contamination.

Cycle 2

Design	UV the laminar hood for 20 minutes, and completely sanitize the insides with 70% ethanol before inoculation and taking each measurement. Repeat the experiment in the same manner as before, taking care to minimize risk of contamination.
Build	Inoculate 3 cultures • KJK01 (without antibiotics) • pCSCX-KJK01 (uninduced + antibiotics) • pCSCX-KJK01 (induced + antibiotics) Take hourly OD readings, store supernatants at -20°C and submit for sucrose assay and NMR analysis.
Test	KJK01 showed growth, while both pCSCX-KJK01 strains did not.
Learn	Suspect contamination in the KJK01 culture again, possibly due to lack of antibiotics. Were advised by mentors to use a different negative control. Decided to replace BG-11 with CoBG-11, as done by Zhang et al, 2020

Cycle 3

Design	Use KJK01 transformed with a blank Kan resistance plasmid (pKD4) as a negative control to show that sucrose consumption is solely due to pCSCX plasmid, and to minimize risk of contamination. Use CoBG-11, a medium containing BG-11 and salts that help E. coli grow better. Repeat experiment with same composition of pCSCX cultures.
Build	Transformation failed, but due to lack of time could not repeat it and chose to inoculate all cultures without antibiotics in order to have a true negative control. Inoculate 3 cultures in CoBG-11 • KJK01 (without antibiotics) • pCSCX-KJK01 (uninduced + antibiotics) • pCSCX-KJK01 (induced + without antibiotics) Take hourly OD readings, store supernatants at -20°C and submit for sucrose assay and NMR analysis.
Test	KJK01 and pCSCX-KJK01 (induced) showed growth, while pCSCX-KJK01 (uninduced) did not. Cannot confirm if growth is of our strains or the contaminants due to lack of antibiotics in the medium. Samples have been submitted for sucrose assay and NMR analysis, however results have not arrived yet.
Learn	Will perform site directed mutagenesis on pCSCX plasmid to render cscABK genes non-functional, and transform KJK01 with it. This will serve as a true negative control for the next assay. CoBG-11 will be used next time as well. Cultures might not have grown due to low concentrations of sucrose having been added. Will repeat with higher concentrations of sucrose, look at Results.

References

Zhang, L., Chen, L., Diao, J., Song, X., Shi, M., & Zhang, W. (2020). Construction and analysis of an artificial consortium based on the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 to produce the platform chemical 3-hydroxypropionic acid from CO 2. Biotechnology for biofuels, 13(1), 1-14.

Sucrose Assay

For sucrose quantification, we used the Megazyme Sucrose/D-Glucose K-SUCGL kit¹. The kit uses the enzymes glucose oxidase, peroxidase and fructosidase (invertase).

Sucrose is hydrolyzed by invertase to D-glucose and D-fructose.

The D-glucose content is determined by conversion to a red-colored quinoneimine dye (by the action of glucose oxidase, peroxidase) and the D-glucose concentration is quantified via absorbance measurements at 510 nm.

For every sample, two absorbance measurements must be taken - one of the sample without beta-fructosidase, denoted as solution A, to measure the initial amount of free D-glucose present in the sample, and one of the sample with beta-fructosidase, denoted as solution B, to measure the final amount D-glucose in the sample (this includes any initial amounts of free D-glucose, and any sucrose that got converted to D-glucose by the action of beta-fructosidase). The difference in the absorbance between these two solutions gives the amount of sucrose in the sample.

The kit requires the sample to contain a sucrose concentration between 0.02 g/L and 0.5 g/L, for which it prescribes dilution instructions based on the estimated amount of sucrose and glucose in the sample.

However, since we are dealing with bacterial culture supernatants containing unknown sucrose concentrations, we would not know how much to dilute it by. In this interest, we decided to plot a standard curve of absorbance vs sucrose concentration.

The absorbance readings increase linearly with sucrose concentration over a range of concentrations, before plateauing out at some value. If a particular sample yields an absorbance in the plateau range, it needs to be diluted down into the linear range before it can be measured again.

Cycle 1

Design	Verify if the Megazyme sucrose D-glucose colorimetric assay kit works with sucrose solutions of known concentration
Build	Prepared sucrose solutions of known concentrations 0.1 g/L and 10 g/L Used Megazyme’s kit protocol to verify the concentrations
Test	Solution A gave negative absorbance readings compared to the distilled water blank for both samples instead of the expected absorbance of 0. Received large error in calculated concentration relative to the prepared concentrations.
Learn	Since both samples were diluted to the same concentration as per the kit’s protocol, they should have given the same absorbance reading for Solution B, which they failed to do so. Instead of distilled water as a blank, use distilled water + acetate buffer as blank for solution A, and distilled water + beta-fructosidase as blank for solution B. The biospectrometer may be faulty, use a 96 well plate reader instead. Prepare fresh samples of sucrose solutions.

Cycle 2

Design	Prepare a standard curve of sucrose concentration versus absorbance. Use the curve to estimate concentration of sucrose from unknown samples/bacterial culture supernatants.
Build	Prepared fresh sucrose solutions of known concentrations: 0, 0.1, 2.5, 5, 7.5, 10, 15, 20 g/L Performed the enzymatic reactions as per the kit protocol and measured absorbance in a 96 well plate reader. Plotted absorbance of the samples against their known concentrations.
Test	Obtained no discernible trend.
Learn	Re-prepare sucrose samples with more precision and redo the standard curve. Store sucrose solution samples in the refrigerator to prevent any contamination.

Cycle 3

Design	Re-prepared fresh sucrose samples to construct a standard curve.
Build	Prepared fresh sucrose solutions of known concentrations: 0, 0.01, 0.05, 0.5, 1, 2.5, 5, 10, 20 g/L Performed the enzymatic reactions as per the kit protocol and measured absorbance in a 96 well plate reader.
Test	Plotted absorbance of the samples against their known concentrations. Obtained a curve that follows a linear trend at concentrations in the range 0 to 2.5 g/L and then begins to plateau. The relative error between the calculated concentration of the samples and their known concentrations was fairly constant in the concentration range between 0.05 g/L and 1 g/L.
Learn	The linear concentration range we obtained corresponds to the kit’s prescribed sucrose concentration range in which prior dilution is unnecessary. Thus, we have determined the sucrose concentration range in which the concentration can be measured with minimal and constant error - 0.05 to 1 g/L

References

https://www.megazyme.com/sucrose-d-glucose-assay-kit

S. elongatus growth curves

Cycle 1

Design	Acclimate S. elongatus UTEX 2973-WT and UTEX 2973-cscB to 150 mM NaCl
Build	Inoculate WT and cscB cultures in BG11 with 150 mM NaCl and grow overnight until the culture is lime green and reaches an OD greater than 0.8
Test	Culture grows very slowly, does not grow healthy overnight
Learn	Cultures pause their growth under salt stress for an acclimatory period.

Cycle 2

Design	Continue acclimation till maximum OD and inoculate new 100 mL cultures with 150 mM NaCl of OD in range 0.1 to 0.2
Build	cscB reached an OD of 0.6, while WT crossed 0.8. The former was pelleted down and inoculated into a new culture, while the latter could be transferred directly without pelleting. Two replicates of each were maintained
Test	The cscB cultures entered stasis and took a while to grow, while the WT cultures temporarily decreased in OD.
Learn	Maintain similar treatment for both cultures to have comparable initial ODs.

Cycle 3

Design	Take measurements of OD and supernatant concentration of sucrose every three hours
Build	2 1mL-aliquots were taken from each culture for each measurement. The absorbance at 730 nm was measured for one aliquot. The other one was centrifuged to separate and store the supernatant for sucrose assay use.
Test	The growth rates were stunningly slow. Fitting the data to a logistic curve, we got doubling times of 27 and 36 hours in the exponential phase(given by T_double=ln(2/r)). This is much slower than the minimum according to the literature in the range of 2 hours in BG11 medium at 41°C under continuous 500 μmoles photons·m^-2·s^-1 white light with 3% CO₂¹. The sucrose produced by the cscB strain was 0.13 g/L while the literature reports a yield of 5 g/L in the same time.
Learn	We decided to optimise the growth conditions for the culture to high carbon (0.5-0.6% as opposed to 0.04% of ambient CO2) and high light (150 μEm^-2s^-1).

Cycle 4

Design	Prepare more BG-11 and then add bicarbonate to the medium
Build	We made the medium and autoclaved it
Test	The medium did not have a steady pH, crossing beyond 8.3 into 9+
Learn	We need to stabilize the pH with TES buffer (as recommended by Dr. Michelle Liberton). As the CoBG11 medium optimized for co-culture with E. coli by Zhang et al, 2020² includes the TES buffer, 150 mM of NaCl, and some additional NH₄Cl, we decided to use it for the OD curve.

Cycle 5

Design	We repeat the WT and cscB growth curves in CoBG11 with bicarbonate, controlling for the presence or absence of IPTG
Build	Acclimated the two strains, then inoculated one WT culture in 100mL CoBG11, along with 4 cscB cultures, of which two were induced with IPTG
Test	The cultures grew very strangely, growing greener and duller in unpredictable ways, perhaps because we have not optimised the frequency of bicarbonate flashing. However, the OD increased faster, reaching 1.8 in the case of one of the uninduced cscB cultures. At one point, the cells aggregated in the cscB cultures when they were removed from the incubator for a measurement, but the aggregates later disappeared. The sucrose assays for these were ongoing as of the wiki freeze.
Learn	We need to optimise the bicarbonate flashing frequency.

References

Yu, J., Liberton, M., Cliften, P. F., Head, R. D., Jacobs, J. M., Smith, R. D., ... & Pakrasi, H. B. (2015). Synechococcus elongatus UTEX 2973, a fast growing cyanobacterial chassis for biosynthesis using light and CO 2. Scientific reports, 5(1), 1-10.
Zhang, L., Chen, L., Diao, J., Song, X., Shi, M., & Zhang, W. (2020). Construction and analysis of an artificial consortium based on the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 to produce the platform chemical 3-hydroxypropionic acid from CO 2. Biotechnology for biofuels, 13(1), 1-14.

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Co-culture modeling

Cycle 1

Cycle 1

Design	The design includes two modeling techniques - dynamic and steady state models, within which similar parameters could be varied and their effects could be analyzed.
Build	Steady state model - A joint model of the co-culture was built using the individual genome-scale models of S. elongatus and E. coli. Dynamic model - built by adapting the dynamic model made by the Toulouse INSA-UPS team.
Test	Varying parameters such as sucrose productivity of S. elongatus, butanol productivity of E. coli, O2 level in the consortium and CO2 uptake, to see its effects on the butanol yield and the population dynamics of the organisms.
Learn	The two models, based on different assumptions, provide similar results on strategies to optimize butanol production by the co-culture.

Building the SteadyCom model

Cycle 1
Cycle 2

Cycle 1

Design	The steady state model was designed to be built using the SteadyCom algorithm. This requires making a joint model of certain specifications built from the individual genome scale models.
Build	The joint model was built using CreateMultispeciesmodel.m function in the COBRA Toolbox from modified versions of iML1515 and iSyu683.
Test	SteadyCom was run on the joint model. However, the results showed that the co-culture was infeasible.
Learn	To troubleshoot and understand the specific issue for this infeasibility, cultures of just E. coli and S. elongatus were individually built with CreatMultispeciesmodel.m function and analysed with SteadyCom. Results showed that the issue was in the cyanobacteria model due to lack of standardization of reaction names and the format of sink reactions. This prevented the CreateMultispeciesmodel.m function from identifying the reactions correctly.

Cycle 2

Design	Reaction names were changed in the genome scale model and were manually curated into the joint model by us.
Build	SteadyCom was run using the new joint model.
Test	SteadyCom showed a successful steady state growth of both organisms with the production of butanol.
Learn	Sink reactions in the individual species that take metabolites from the intracellular compartments are not recognized by the CreateMultispeciesModel function and hence not included in the joint model and have to be manually added.

Computational - Experimental Integrated Engineering Cycle

Notation:

We use the following shorthand terms:

By sucrose productivity we denote the rate of sucrose production from S. elongatus in mmol gDW^-1 hr^-1
By butanol productivity we denote the rate of butanol production from E. coli KJK01 and its transformants in mmol gDW^-1 hr^-1.

Cycle 1

Design	Based on the shortlisted overexpression and deletion targets from our strain optimization modeling, we will design experiments to actually perform these modifications in S. elongatus and E. coli.
Build	We will then execute these experiments and measure the sucrose and butanol productivity of all the modified strains
Test	We will compare this with the original strains to see if the modifications have made a significant difference to strain productivity.
Learn	The modified strains which give the maximal sucrose and butanol productivity will be selected to be used in the future for the co-culture.

Cycle 2

Design	Our co-culture modeling predicted that increasing both the butanol productivity of E. coli and the sucrose productivity of S. elongatus will increase the total butanol yield from the co-culture. However, this must be experimentally tested. We will design experiments to compare the butanol yield of the co-culture made with the optimized strains from the previous iteration with the butanol yields of the co-culture made with the unoptimized strains.
Build	We will execute these experiments and measure the butanol yield from both co-cultures.
Test	We will compare the butanol yields and see if using the optimized strains with higher sucrose and butanol productivity gives a higher butanol yield in co-culture.
Learn	Based on the results, we will know if the results of our co-culture modeling are valid and whether increasing sucrose and butanol productivity in fact does increase the co-culture butanol yield

Cycle 3

Design	Our co-culture modeling also predicted that extracting oxygen from the co-culture would increase butanol yield. These results must also be experimentally tested. We will design experiments to test the butanol yield from the co-culture at different oxygen levels.
Build	We will execute these experiments and measure the butanol yields at varying oxygen levels.
Test	We will identify the oxygen level at which butanol yield is maximal.
Learn	Based on the above, we will know if the result from our co-culture modeling that a microaerobic setting for E. coli will give us the highest butanol yield is valid.