# Team:IISER-Pune-India/Results

<!DOCTYPE html> Results

# PCR and gel electrophoresis for confirmation of butanol producing genes

To confirm whether the E. coli KJK01 strain contained the butanol producing genes, we extracted the genomic DNA of the cells and subjected it to PCR amplification using primers we received from Dr Yazdani’s lab. Since the cassette has been added as one single component, verification of any component from the cassette will verify the presence of the butanol producing genes in KJK01[1]. We received primers against the hbd-crt genes. An amplicon of the length of ~750bp from the PCR would verify the presence of those genes.

## Genomic DNA extraction

#### Expected Result:

Large band above the DNA ladder, as gDNA is very large in size and cannot traverse through the gel

After the second genomic DNA extraction attempt, when we ran it on a gel, we obtained a single band above the DNA ladder along with a large bright band at the bottom of the ladder. We checked the concentration of the extracted genomic DNA using a NanoDrop and observed a concentration of 2.9 μg/mL.

#### Result:

• In the first genomic DNA extraction attempt, we obtained fragmented DNA which appeared as multiple bands when we loaded the gDNA on a gel.
• The second genomic DNA extraction yielded a single band above the DNA ladder along with a large bright band at the bottom of the ladder
• The first band is the gDNA of KJK01 and the bright band at the bottom might be RNA contamination due to improper addition of RNase.

Gel showing gDNA band above DNA ladder. Identical samples loaded next to ladder. Bright band at the bottom might be RNA contamination.

## PCR amplification of hbd-crt cassette and gel electrophoresis

#### Expected Results:

Amplicon of length ~750bp showing up on the gel

#### Results:

PCR with gDNA extract 1:

When PCR was performed using the first gDNA extract as a template, we observed no bands on running the PCR product on a gel.

Failed PCR gel

## Troubleshooting for PCR with gDNA extract 1

...

Despite running the gel multiple times, we failed to obtain a positive result. The modifications we made to our protocol to get the desired results were -

• Reduce dNTP: Our mentor advised us to reduce the amount of dNTP added because the amount could be deleterious to the reaction. The concentration was reduced to 100-500 ng/µl.
• Reduce gDNA concentration: With the same logic as of dNTP, genomic DNA concentration was reduced to 100 ng/µl.
• Compare primer with negative control: To find whether the primers were binding rightly or non-specifically or binding at all, a test was compared with negative control wherein the negative control was devoid of just the gDNA. If the primer dimer band has reduced intensity in the test, it means that the primer is binding to the DNA. If the band is not of the right size, then it is binding non-specifically.

But we were unable to see the primer dimer bands at all in the test lanes.

Check quality of gDNA:

We ran the gDNA extract 1 on a gel to find that it had fragmented, and so we performed the extraction again with a modified protocol - we suspected that the elution buffer used to resuspend the gDNA in the first attempt was contaminated by nuclases and so we used autoclaved ddH2O (MilliQ) the second time around.

PCR with gDNA extract 2:

No bands appeared on imaging the PCR product.

As none of our troubleshooting worked, we spoke to our PI, Prof Pucadyil, from whose lab we borrowed the Pfu polymerase used in the PCR reaction. He pointed out that we hadn’t used DMSO in any of the previous runs when the protocol especially called for it. We had been following the PCR protocol of another lab. He suggested that we perform a gradient PCR at different concentrations of DMSO and different annealing temperatures.

Our mentor, Trisha suggested that the potential RNA contamination seen in the second gDNA extraction attempt might have led to an overestimate in the NanoDrop’s estimation of the concentration of the gDNA. She advised us not to dilute the gDNA template before adding it to the PCR mix for gradient PCR.

We conducted 20 PCR reactions in parallel under the following conditions:

Conditions
Negative Control
0% DMSO
5% DMSO
10% DMSO
50°C
Gel 1
Gel 1
Gel 1
Gel 1
52°C
Gel 2
Gel 2
Gel 2
Gel 2
54°C
Gel 2
Gel 2
Gel 2
Gel 2
56°C
Gel 2
Gel 2
Gel 2
Gel 2
60°C
Gel 2
Gel 2
Gel 2
Gel 2

#### Result:

• Bands found at ~750bp in the 5% and 10% DMSO bands
• Gel 2 was left on for too long and ran out.

Gel 1. Bright bands seen at ~750 bp in the 5% and 10% DMSO lanes. No band seen in negative control and 0% DMSO lanes.

This confirmed the presence of butanol producing genes in KJK01.

# NMR confirmation of butanol production

As PCR confirmation of the presence of the butanol producing genes was taking too long, we simultaneously started culturing KJK01 in Luria–Bertani (LB) broth as well as Terrific Broth (TB). Abdelaal[1] et al cultured the strain in TB as E. coli has been shown to produce maximum butanol in that medium. Making TB is a rather time-consuming process for us, hence we decided to check if we could perform the rest of the quantification of butanol production in LB cultures.

We inoculated 6 2&#176 cultures as follows:

• LB (0 IPTG)
• LB (0.1 mM IPTG)
• LB (0.2 mM IPTG)
• TB (0 IPTG)
• TB (0.1 mM IPTG)
• TB (0.2 mM IPTG)

#### Expected results:

The TB cultures are expected to produce more butanol than the LB cultures. The IPTG induced cultures are expected to produce more butanol than the ones with 0 IPTG.

#### Results:

Sample
Butanol relative intensity
Standard relative intensity
Concentration of butanol (μM)
TB no IPTG
2.189
0.6995
208.6356397
TB 0.1
4.379
0.5964
489.5169852
TB 0.2
3.972
0.5579
474.6607636
LB no IPTG
1.6
0.883
120.806342
LB 0.1
15.14
1.157
872.4146932
LB 0.2
12.8
1.14
748.5754386

NMR spectrum for LB 2&#176 cultures

Characteristic butanol peaks are amplified in 0.1 mM (red) and 0.2 mM (green) IPTG induced cultures

NMR spectrum for TB 2&#176 cultures

Characteristic butanol peaks are amplified in 0.1 mM (red) and 0.2 mM (green) IPTG induced cultures

• The spectrum contains peaks characteristic of butanol, which indicates that butanol is present in the supernatant and produced by KJK01
• The LB culture produced ~1.5 times the amount of butanol produced by the TB cultures
• The supernatants of cultures that were not induced with IPTG showed little butanol content, suggesting leaky expression of the promoter the genes are under
• The supernatants of cultures induced with 0.1 mM IPTG contained slightly more butanol than the ones induced with 0.2 mM IPTG
• KJK01 has been shown to produce equal amounts of ethanol, but due to time constraints we could not analyse the spectrum for ethanol peaks

# Transformation of KJK01 with pCSCX plasmid

KJK01 was transformed with the pCSCX plasmid that would enable it to survive with sucrose as its sole carbon source.[3] The cells were transformed via electroporation and transformed cells were streaked onto LB agar plates containing Kanamycin and Ampicillin.

#### Results:

After overnight incubation, many colonies were seen on the plates, indicating that transformation was successful. Glycerol stocks were made from these plates immediately.

LB agar + Kan50 + Amp100 plates showing colonies of transformants

# Growth curve assay for KJK01 and pCSCX-KJK01

We aimed to grow KJK01 and pCSCX-KJK01 in LB and characterize their growth. Due to lack of time in the lab in accordance with COVID-19 restrictions in India, we could perform only one assay and hence could not calculate the growth rates.

#### Expected Result:

We expect a sigmoid growth curve for both strains. pCSCX-KJK01 is expected to grow slower than KJK01 due to the metabolic burden of carrying an extra plasmid.

#### Results:

Growth curve of KJK01 2&#176 culture in LB over 11 hours

Growth curve of pCSCX KJK01 2&#176 culture in LB over 11 hours

• Both strains grow much slower than WT MG1655, probably due to all the modifications made to their genome
• pCSCX-KJK01 grew slower than KJK01, as expected
• When we fit the data to a logistic growth curve, governed by the equation $${dN \over dt} = {Nr(1 - {N \over k})}$$, we found that the difference in the inherent growth rate, $$r$$,is small: E. coli KJK01 grew at a rate of 0.680 whereas E. coli pCSCX-KJK01 grew at a rate of 0.596.
• However, the carrying capacities differed greatly: $$K_{KJK01}$$ was around 6.45 while $$K_{pCSCX-KJK01}$$ was only 3.44.
• We may interpret this to mean that the medium is incapable of supporting higher populations of the transformant as compared to the wild type. The growth curve at a particular OD can be seen by looking at the slope of the graph at a particular value on the y-axis.
• The OD600 of the KJK01 strain increased rapidly to 3, above which our spectrophotometer did not show any reading, causing us to dilute our samples to 50% of the original concentration
• In a certain range, the relationship between OD and cell count follows the Beer-Lambert law and is linear, which is why we can double the reading values obtained after dilution[2]
• We decided to stop taking readings when the cultures reached an OD600 of ~6, as our spectrophotometer stopped showing readings again
• The spectrophotometer in our lab does not give accurate readings above OD600 1, which is why readings from our experiment above that limit are not very reliable
• We took 5 readings per sample, but the standard deviation was rather small. This lead to error bars smaller than the point size in our graphs, which is why they are not clearly visible, but are certainly present.

# MIC assay of butanol and ethanol for KJK01

We performed a MIC assay of butanol and ethanol to identify the minimum concentrations of these compounds at which the growth of our strain gets inhibited. We performed the assay in three 96 welled plates. We used the wild type E. coli strain MG1655 as a control. Our concentration range was from 0.24 to 60 mg/mL (9 dilutions).

#### Expected Results

For our wild type strain, we expect a MIC of 7.5 to 15 mg/mL for butanol and above 15 mg/mL for ethanol.[4],[5] Since we have not made any modifications in KJK01 to increase it’s butanol tolerance, we expect similar results in this strain.

#### Results

MIC Graphs for plate 1

MIC Graphs for plate 2

MIC Graphs for plate 3

Strain
Compound
MIC (mg/mL)
MG1655
Butanol
1.87 - 3.75
Our three plates do not agree to a single value however, two of our plates show closeby values.
KJK01 uninduced
Butanol
1.87
We observed contamination in plate 1 for concentrations of 3.75 and 7.5 mg/mL. However, plates 1 and 2 agree on the MIC. The third plate is an outlier.
KJK01 induced
Butanol
60
We observed a high starting OD in some wells of plate 3. However, 2 of our 3 plates indicate this as our MIC. For confirmation, we will have to perform the MIC assay with a higher concentration range.
MG1655
Ethanol
60
All our plates indicate this as the MIC. We can further refine this by considering a different concentration range.
KJK01 uninduced
Ethanol
30
We observed contamination of a few wells in plate 3 contamination. However, our other two plates are suggestive of an MIC of 30 mg/mL
KJK01 induced
Ethanol
30
Two of our three plates agree on this value.

Our results fall close to the range we expected. We observed low tolerance of our strains to butanol. Our induced strain seemed to show a higher tolerance to butanol than the others which suggests that the genes that produce butanol also help tolerate it. Whatever may be the mechanistic reason, the high tolerance of our butanol producing strain works in our favour.

The ethanol tolerance of our strains is as we had predicted. High ethanol tolerance is advantageous for us since our strain produces equal amounts of butanol and ethanol.

# Sucrose assay for KJK01 in LB

Three strains of E. coli were grown in LB medium to check their sucrose consumption rate. Using the Megazyme Sucrose/D-Glucose Assay Kit,[6] we were able to quantify the amount of sucrose left in our medium over time. This helped us analyse how much sucrose was being taken up by our culture over a period of 24 hrs. Initial concentration of sucrose supplied to the media was 0.25 g/L.

The strains under study were

• KJK01 (negative control)
• pCSCX-KJK01 + antibiotics (uninduced)
• pCSCX-KJK01 + antibiotics + 0.1 mM IPTG (induced)

#### Expected Results

• KJK01 was expected to consume minimal/no sucrose
• pCSCX-KJK01, both induced and uninduced, were expected to consume sucrose

#### Results

• The E. coli KJK01 consumed most sucrose. Out of the 0.25g/L sucrose which was supplied to the media, only 0.006g/L was left after a period of 24 hours, confirming that the culture had consumed it almost entirely.
• We however cannot confirm if the sucrose was consumed by KJK01 or other contaminants, due to lack of antibiotics in the medium
• The results for pCSCx-KJK01, induced and uninduced, could not be calculated as our spectrophotometer readings were not reliable

Gradual decrease in the red colour of quinoneimine dye shows decreasing sucrose concentration in the media over time.

Decreasing sucrose content in the media of wild type E. coli KJK01 over a period of 24 hours.

### References

1. Ali Samy Abdelaal, Kamran Jawed, Syed Shams Yazdani, CRISPR/Cas9-mediated engineering of Escherichia coli for n-butanol production from xylose in defined medium, Journal of Industrial Microbiology and Biotechnology, Volume 46, Issue 7, 1 July 2019, Pages 965–975, https://doi.org/10.1007/s10295-019-02180-8
2. Stevenson, K., McVey, A. F., Clark, I. B., Swain, P. S., & Pilizota, T. (2016). General calibration of microbial growth in microplate readers. Scientific reports, 6(1), 1-7. https://doi.org/10.1038/srep38828
3. Bruschi, M., Boyes, S. J., Sugiarto, H., Nielsen, L. K., & Vickers, C. E. (2012). A transferable sucrose utilization approach for non-sucrose-utilizing Escherichia coli strains. Biotechnology advances, 30(5), 1001–1010. https://doi.org/10.1016/j.biotechadv.2011.08.019
4. Wilbanks, B., Trinh, C.T. Comprehensive characterization of toxicity of fermentative metabolites on microbial growth. Biotechnol Biofuels 10, 262 (2017). https://doi.org/10.1186/s13068-017-0952-4
5. Knoshaug, E. P., & Zhang, M. (2009). Butanol tolerance in a selection of microorganisms. Applied biochemistry and biotechnology, 153(1-3), 13–20. https://doi.org/10.1007/s12010-008-8460-4
6. https://www.megazyme.com/sucrose-d-glucose-assay-kit

# Growth curve - Ambient carbon dioxide

We aimed to characterize the growth of our S. elongatus UTEX 2973 wild type and engineered strain. The population density of our culture is measured in terms of the optical density/absorbance of the culture measured at 730 nm (a wavelength at which there is no absorbance due to photosynthetic or other pigments)[1]. After setting up the culture, a 1 mL sample was taken every 3 hours and its OD was measured in a biospectrometer.

We carried out this experiment with two replicates of WT and two of the cscB-incorporated strain. The data were fit to a logistic growth curve, and $$K-$$ and $$r-$$ values were estimated.

Recall that logistic growth is defined by the equation:

$${dN\over dt} = Nr (1 - {N \over K})$$

Which can be integrated to yield

$$N_t = {K \over {1 + Ae^{-rt}}}$$ where $$A = { N_0 \over {K - N_0}}$$

Standard deviation was estimated from the difference between the mean of the two replicates and either value; a smaller source of error was the instrumental error, which was added in quadrature as a correction to the former estimate.

At OD730 values above 0.6, we began seeing fluctuations in the second decimal digit of our biophotometer reading. As these were larger fluctuations, we began diluting the samples for measurements in the more precise range and calculating the OD of the original culture from our measured value.

The first iteration of this assay was carried out at ambient carbon dioxide levels - that is, 0.04%. We did not induce the gene cscB with IPTG, nor did we use high light levels. The growth curves resulting from these experiments are provided below:

• S. elongatus UTEX 2973-WT

• S. elongatus UTEX 2973-cscB

As we’ve fitted the data to a logistic model, we cannot speak of a single “doubling time” for a UTEX 2973 culture - the rate of doubling would decrease as the culture approaches its carrying capacity. However, at low ODs, the curve is effectively identical to an exponential graph, and the doubling time can be estimated in that neighborhood, as given by the formula:

$$T_{double} = {ln(2) \over r}$$

These doubling times are incredibly slow - 27 hours for the wild type and 63 hours for the cscB-transformant. We encourage you to verify this on the graphs above! According to the literature, S. elongatus UTEX 2973 can be grown at doubling times shorter than two hours![2]

It is relevant to note that the $$K$$-value for the cscB strain is essentially infinite - in other words, the curve is indistinguishable from an exponential growth curve.

# Sucrose Assay

We used Megazyme’s colorimetric assay to estimate sucrose concentration.[3] The assay kit involves hydrolyzing sucrose to produce glucose and fructose, oxidizing the glucose to produce peroxide, and quantifying the peroxide colorimetrically (which is used up to produce a quinoneimine fluorescent dye). We first prepared standard graphs of absorbance versus sucrose concentration, to identify a range of values within which the two bear a linear relationship. We then used the slope of this curve to estimate the sucrose concentration in our ambient-carbon cultures of S. elongatus UTEX 2973 grown at a light intensity of ~100 mol photons/m2/s. 1mL supernatant samples of the culture were taken and stored at 4℃.

In accordance with theory, the wild type produced no sucrose. The cscB strain, however, did produce some sucrose over time. We obtained a maximum titer of 0.09 g/L of sucrose in our cscB strain grown in BG-11, at ambient carbon dioxide levels, without any IPTG induction, over a period of 60 hours. This is significantly lower than the titer obtained with the same strains in Lin et al. (2020), which was around 5 g/L over 72 hours.[4] This is unsurprising, as that study took place under vastly different conditions - the strains were grown at a CO2 concentration of 0.5-0.6%, at a light intensity of 250 mol photons/m2/s, and were induced by 1 mM of IPTG.

# Growth curve - High carbon dioxide

Next, we grew the cscB expressing strain of UTEX 2973 again while replicating the conditions from Lin et al. (2020), to analyze its growth and sucrose production. Both induced (1mM IPTG) and uninduced (0mM IPTG) cultures were maintained in two replicates each. Measurements were taken every 12 hours, and the standard deviation for each data point was calculated as previously.

However, since our incubator is not equipped to supply and maintain CO2, we resorted to bicarbonate flashing: an inexact technique of simulating higher carbon dioxide concentrations by regular manual addition of an equivalent quantity of bicarbonate ions into the medium[5]. We did not have time to optimise the frequency of bicarbonate flashing, or to experiment with refurbishing the nutrients in the medium. We think it is for these reasons that the cultures frequently turned dull and occasionally crashed in OD.

The noise due to these large fluctuations in OD has severely impacted the goodness-of-fit of our logistic curves. These poor fits are provided below:

• WT (negative control)

• cscB(IPTG+)

• cscB(IPTG-)

One insight from these curves is that growth at low ODs is indeed much higher under high carbon and high light settings - the doubling times in the exponential phase were now 6.9 hrs, 23 hrs and 13.9 hrs for the WT, cscB (IPTG+) and cscB (IPTG-) respectively. The lack of steady growth is concerning and we would like to try and optimise the frequency of butanol flashing to get steadily growing cultures.

Unfortunately, we did not have the time to run the sucrose assay on the supernatants we collected under high carbon and high light conditions as of the Wiki freeze. We expect to see a rise in the sucrose titer as a consequence of the increased carbon flux in the system.

### References

1. Chioccioli, M., Hankamer, B., & Ross, I. L. (2014). Flow cytometry pulse width data enables rapid and sensitive estimation of biomass dry weight in the microalgae Chlamydomonas reinhardtii and Chlorella vulgaris. PLoS One, 9(5), e97269.
2. 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.
3. https://www.megazyme.com/sucrose-d-glucose-assay-kit
4. Lin, PC., Zhang, F. & Pakrasi, H.B. Enhanced production of sucrose in the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973. Sci Rep 10, 390 (2020). https://doi.org/10.1038/s41598-019-57319-5
5. Sengupta, A., Pritam, P., Jaiswal, D., Bandyopadhyay, A., Pakrasi, H. B., & Wangikar, P. P. (2020). Photosynthetic Co-Production of Succinate and Ethylene in A Fast-Growing Cyanobacterium, Synechococcus elongatus PCC 11801. Metabolites, 10(6), 250.

# Growth curve assay for KJK01 and pCSCX-KJK01 in BG-11 + sucrose

We aimed to grow KJK01 and pCSCX-KJK01 in BG-11 + sucrose and characterize their growth. BG-11 does not have any carbon source[1] as it is meant for photosynthetic organisms. pCSCX is a plasmid containing the cscABK cluster that helps in the transport and metabolism of sucrose[2]. In the mixture of BG-11 + sucrose, sucrose is the sole carbon source for E. coli. If the strain manages to grow in this mixture, it means that it has the genes necessary for sucrose consumption.

This experiment served as a proxy for our co-culture, as it indicated that our E. coli strains were able to subsist on plain sucrose. The next step would be to grow the strains in BG-11 + supernatant of the cyanobacterial culture in order to see how the additional compounds secreted by S. elongatus affect the growth of the E. coli, and finally moving on actually culturing the two organisms together.

We inoculated 3 cultures:

• KJK01 (negative control)
• pCSCX-KJK01 + antibiotics (uninduced)
• pCSCX-KJK01 + antibiotics + 0.1 mM IPTG (induced)

The supernatants would simultaneously be used for sucrose consumption and NMR quantification assays as well, to obtain sucrose consumption rates and butanol production rates for the three cultures.

#### Expected Results:

• KJK01 (negative control) - This culture is expected to show negligible/no growth and no butanol production.
• pCSCX-KJK01 (uninduced) - This culture is expected to show fastest growth and low butanol production.
• pCSCX-KJK01 (induced with 0.1 mM IPTG) - This culture is expected to show slow growth and maximum butanol production.

#### Results:

We repeated this experiment twice and obtained two growth curves for each of the three strains.

Growth Curve 1:

KJK01 growth curve in BG-11 + sucrose over 24 hours

pCSCX KJK01 (uninduced) growth curve in BG-11 + sucrose over 24 hours

pCSCX KJK01 (induced) growth curve in BG-11 + sucrose over 24 hours

• KJK01 - This culture showed good growth, reaching OD600 0.3 in 24 hours
• pCSCX-KJK01 (uninduced) - This culture showed no growth in 24 hours
• pCSCX-KJK01 (induced with 0.1 mM IPTG) - This culture showed no growth in 24 hours

This was a rather unexpected result and the complete opposite of what we thought would happen. We suspected that the first culture might have been contaminated, and the contaminants were able to survive on sucrose. We could have confirmed if the KJK01 was growing if we had submitted our supernatants for NMR analysis, but the slots were already full.

Growth Curve 2:

We repeated the same experiment as in growth curve 1, but took readings every two hours instead of one hour, as taking 6 samples, measuring their OD, centrifuging them and freezing the supernatants every hour was simply not practical. We made sure to UV the hood and clean it thoroughly with 70% ethanol in order to minimize chances of contaminating the wild type culture which did not have any antibiotics.

KJK01 growth curve in BG-11 + sucrose over 40 hours

pCSCX KJK01 (uninduced) growth curve in BG-11 + sucrose over 40 hours

pCSCX KJK01 (induced) growth curve in BG-11 + sucrose over 40 hours

• KJK01 - This culture showed good growth, reaching OD600 0.17 in 40 hours
• pCSCX-KJK01 (uninduced) - This culture showed no growth in 40 hours
• pCSCX-KJK01 (induced with 0.1 mM IPTG) - This culture showed no growth in 40 hours

# Transformation of KJK01 with pKD4 plasmid

KJK01 was transformed with the pKD4 plasmid, which is a blank plasmid containing only Kanamycin resistance. This strain would serve as a negative control for our next growth curve, and would show that any sucrose consuming properties that have been conferred upon the KJK01 strain is solely via the pCSCX plasmid. The controls in the previous growth curves did not have the same conditions as the rest of the experiment, and hence could not be called true controls. We chose Kan because the sucrose exporting strains of S. elongatus from Prof Pakrasi’s lab have Kanamycin and Gentamycin resistance [3]

#### Results

Transformation failed, we did not see any colonies on the LB agar + Kan50 plates.

A possible reason for this might be because the pKD4 plasmid has an incompatible ori. We were already warned by Dr. Nishad Matange (who lent us the plasmid) that we may not be able to get colonies with pKD4 because it contains oriR𝛄 which requires that require the trans-acting Π protein (the pir gene product) for replication[4].

Dr. Gayatri Panaghat gave an alternative to this by suggesting that we perform site directed mutagenesis on our pcscX plasmid and remove the genes coding for cscABK and replace it with a Kan resistance gene, which would serve as a true negative control. However, we did not have the necessary time for ordering primers and carrying out the mutagenesis process. Hence, we proceeded by Dr Pananghat’s second advice, which was to repeat the experiment with a strict check on possible sources of contamination.

# Growth curve assay for KJK01 and pCSCX-KJK01 in CoBG-11 + sucrose

After our first two growth curves failed, we went back to the Zhang et al[5] paper to look at their protocol, because we did not have time to optimize our current protocol further. We replaced BG-11 with CoBG-11, a medium created by the authors that contains BG-11 as well as certain salts that help E. coli grow better.[5] We also took readings every 6 hours, as the strains seemed to be slow-growing.

We inoculated 3 cultures:

• KJK01 (negative control)
• pCSCX-KJK01 (uninduced)
• pCSCX-KJK01 + 0.1 mM IPTG (induced)

Since the transformation with pKD4 failed, we did not have a valid negative control anymore, hence upon our mentor’s recommendation, we inoculated all cultures without antibiotics.

KJK01 growth curve in CoBG-11 + sucrose over 96 hours

pCSCX KJK01 (uninduced) growth curve in CoBG-11 + sucrose over 96 hours

pCSCX KJK01 (induced) growth curve in CoBG-11 + sucrose over 96 hours

#### Results

• KJK01 - The culture hit an OD600 of 0.26 in 4 days
• pCSCX-KJK01 (uninduced) - The culture showed no growth in 4 days
• pCSCX-KJK01 + 0.1 mM IPTG (induced) - The culture hit an OD600 of 0.22 in 4 days

We cannot tell if the OD is due to contamination or the growth of the culture itself because once again the WT KJK01, which has no sucrose metabolising genes, has managed to grow while the pCSCX-KJK01 (uninduced) culture has not grown. We do however see growth in the pCSCX-KJK01 (induced) culture.

The only way we can confirm if the culture has grown is by analyzing the supernatants to check for the presence of butanol. We have submitted the supernatants for analysis, but unfortunately, the results will not be here before the Wiki freeze deadline. Even if butanol is found in the supernatant we cannot be sure if the E. coli has grown on the sucrose in the medium or some other sugars released by the contaminants due to the absence of antibiotics.

As a part of our future work, we plan to repeat this experiment by using the modified pCSCX plasmid described above as our negative control. We also need to check if the sucrose given to the E. coli is of lower concentration than required. The Zhang et al[5] paper used concentrations up to 200 mg/L based on the maximum concentration of sucrose secreted by the S. elongatus, but their E. coli had the cscABK genes integrated into the genome. The strains containing the pCSCX plasmid had been grown on media containing 2% sucrose[2]. Along with the right negative control, we will also repeat this experiment with higher concentrations of sucrose.

# Sucrose assay

Luria broth (LB) is the most commonly used growth medium for E. coli. Considering that we have planned to use BG-11 as our final co-culture media, we decided to grow three strains of E. coli in a medium containing BG-11 + 0.25g/L as a proxy for our co-culture.

Using the Megazyme Sucrose/D-Glucose Assay Kit[6], we had planned to quantify the amount of sucrose left in our medium over time. This would help us analyse how much sucrose was being taken up by our culture over a period of 24 hrs.

#### Expected Results

• KJK01 was expected to consume sucrose with less rate of consumption
• pCSCX-KJK01, both induced and uninduced, were expected to consume sucrose

#### Results for Growth curve 1

• KJK01 had 0.009 g/L sucrose left in the medium, having the highest rate of sucrose consumption.
• pCSCX-KJK01 + antibiotics (uninduced) had 0.169 g/L sucrose left in the medium, having the lowest rate of sucrose consumption.
• pCSCX-KJK01 + antibiotics + 0.1 mM IPTG (induced) had 0.155 g/L sucrose left in the medium

We can confirm that the sucrose in the pCSCX-KJK01 cultures were consumed by the strain as there were antibiotics in the medium. We cannot however say the same for the KJK01 cultures.

The results for Growth curve 2 and 3 are not ready yet

These experiments are just the beginning to confirm that our transformed pCSCX-KJK01 induced strain of E. coli can not only subsist in the BG-11 medium, but can also efficiently consume sucrose present in the medium.

Hopefully, as we troubleshoot more, we wish to confirm without doubt that the sucrose consuming ability of pCSCX-KJK01 is due to pCSCX, as well as improve their sucrose uptake efficiency.

You can find the raw data for our growth curves and MIC assay here

### References

1. https://www.himedialabs.com/TD/M1541.pdf
2. Bruschi, M., Boyes, S. J., Sugiarto, H., Nielsen, L. K., & Vickers, C. E. (2012). A transferable sucrose utilization approach for non-sucrose-utilizing Escherichia coli strains. Biotechnology advances, 30(5), 1001–1010. https://doi.org/10.1016/j.biotechadv.2011.08.019
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