iGEM 2021 — Team IISER-Pune-India

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# Team:IISER-Pune-India/Notebook

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Notebook

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* Team
  + [Members](https://2021.igem.org/Team%3AIISER-Pune-India/Team)
  + [Attributions](https://2021.igem.org/Team%3AIISER-Pune-India/Attributions)
  + [Collaborations](https://2021.igem.org/Team%3AIISER-Pune-India/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3AIISER-Pune-India/Partnership)
  + [Sponsors](https://2021.igem.org/Team%3AIISER-Pune-India/Sponsors)
* Project
  + [Description](https://2021.igem.org/Team%3AIISER-Pune-India/Description)
  + [Design](https://2021.igem.org/Team%3AIISER-Pune-India/Design)
  + [Notebook](https://2021.igem.org/Team%3AIISER-Pune-India/TeamNotebook/Team%20Notebook%2011e9906b405543a29361e38a50c81024)
  + [Contributions](https://2021.igem.org/Team%3AIISER-Pune-India/Contribution)
  + [Engineering Success](https://2021.igem.org/Team%3AIISER-Pune-India/Engineering)
  + [Future Implementation](https://2021.igem.org/Team%3AIISER-Pune-India/Implementation)
  + [Safety](https://2021.igem.org/Team%3AIISER-Pune-India/Safety)
* Wet Lab
  + [Experiments](https://2021.igem.org/Team%3AIISER-Pune-India/Experiments)
  + [Constructs](https://2021.igem.org/Team%3AIISER-Pune-India/Constructs)
  + [Measurement](https://2021.igem.org/Team%3AIISER-Pune-India/Measurement)
  + [Lab Notebook](https://2021.igem.org/Team%3AIISER-Pune-India/LabNotebook)
  + [Results](https://2021.igem.org/Team%3AIISER-Pune-India/Results)
  + [Future Work](https://2021.igem.org/Team%3AIISER-Pune-India/FutureWork)
* Parts
  + [Basic](https://2021.igem.org/Team%3AIISER-Pune-India/BasicParts)
  + [Composite](https://2021.igem.org/Team%3AIISER-Pune-India/CompositeParts)
* [Model](https://2021.igem.org/Team%3AIISER-Pune-India/Model)
* Human Practices
  + [Integrated Human Practices](https://2021.igem.org/Team%3AIISER-Pune-India/Human_Practices)
  + [Inclusivity and Accessibility](https://2021.igem.org/Team%3AIISER-Pune-India/Inclusivity)
  + [Public Engagement and Education](https://2021.igem.org/Team%3AIISER-Pune-India/Communication)
* [Judging Form](https://igem.org/2021_Judging_Form?id=3971)

# Notebook

* [Introduction](#1)
* [Section 2](#2)
* [Section 3](#3)
* [Section 4](#4)
* [Section 5](#5)
* [References](#6)

# Introduction

---

Cyanobacteria is the green element, both figuratively and literally, of our project. Modelling its behaviour both in a pure culture and in the co culture matters a lot to the direction we put our attention to. Weâ€™ve used FBA and MoMA to predict the growth rates and sucrose production rates of cyanobacteria, both with and without salt stress. Dr.Costas Maranas was generous enough to provide the Genome scale metabolic model for Synechococcus elongatus UTEX 2973 from Mueller et al., 2017.

### Modelling Cyanobacteria for optimum sucrose production

The first hurdle we encountered while modelling cyanobacteria was simulating salt stress. Literature says simulating something like salt stress alters a lot of reactions in the organism and not all of them are straight forward or even known, so we had to make a few assumptions to make the model work.

# Section 2

---

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# Introduction

---

Cyanobacteria is the green element, both figuratively and literally, of our project. Modelling its behaviour both in a pure culture and in the co culture matters a lot to the direction we put our attention to. Weâ€™ve used FBA and MoMA to predict the growth rates and sucrose production rates of cyanobacteria, both with and without salt stress. Dr.Costas Maranas was generous enough to provide the Genome scale metabolic model for Synechococcus elongatus UTEX 2973 from Mueller et al., 2017.

### Modelling Cyanobacteria for optimum sucrose production

The first hurdle we encountered while modelling cyanobacteria was simulating salt stress. Literature says simulating something like salt stress alters a lot of reactions in the organism and not all of them are straight forward or even known, so we had to make a few assumptions to make the model work.

# Introduction

---

Cyanobacteria is the green element, both figuratively and literally, of our project. Modelling its behaviour both in a pure culture and in the co culture matters a lot to the direction we put our attention to. Weâ€™ve used FBA and MoMA to predict the growth rates and sucrose production rates of cyanobacteria, both with and without salt stress. Dr.Costas Maranas was generous enough to provide the Genome scale metabolic model for Synechococcus elongatus UTEX 2973 from Mueller et al., 2017.

### Modelling Cyanobacteria for optimum sucrose production

The first hurdle we encountered while modelling cyanobacteria was simulating salt stress. Literature says simulating something like salt stress alters a lot of reactions in the organism and not all of them are straight forward or even known, so we had to make a few assumptions to make the model work.

# Introduction

---

Cyanobacteria is the green element, both figuratively and literally, of our project. Modelling its behaviour both in a pure culture and in the co culture matters a lot to the direction we put our attention to. Weâ€™ve used FBA and MoMA to predict the growth rates and sucrose production rates of cyanobacteria, both with and without salt stress. Dr.Costas Maranas was generous enough to provide the Genome scale metabolic model for Synechococcus elongatus UTEX 2973 from Mueller et al., 2017.

### Modelling Cyanobacteria for optimum sucrose production

The first hurdle we encountered while modelling cyanobacteria was simulating salt stress. Literature says simulating something like salt stress alters a lot of reactions in the organism and not all of them are straight forward or even known, so we had to make a few assumptions to make the model work.

# Introduction

---

Cyanobacteria is the green element, both figuratively and literally, of our project. Modelling its behaviour both in a pure culture and in the co culture matters a lot to the direction we put our attention to. Weâ€™ve used FBA and MoMA to predict the growth rates and sucrose production rates of cyanobacteria, both with and without salt stress. Dr.Costas Maranas was generous enough to provide the Genome scale metabolic model for Synechococcus elongatus UTEX 2973 from Mueller et al., 2017.

### Modelling Cyanobacteria for optimum sucrose production

The first hurdle we encountered while modelling cyanobacteria was simulating salt stress. Literature says simulating something like salt stress alters a lot of reactions in the organism and not all of them are straight forward or even known, so we had to make a few assumptions to make the model work.

# Introduction

---

Cyanobacteria is the green element, both figuratively and literally, of our project. Modelling its behaviour both in a pure culture and in the co culture matters a lot to the direction we put our attention to. Weâ€™ve used FBA and MoMA to predict the growth rates and sucrose production rates of cyanobacteria, both with and without salt stress. Dr.Costas Maranas was generous enough to provide the Genome scale metabolic model for Synechococcus elongatus UTEX 2973 from Mueller et al., 2017.

### Modelling Cyanobacteria for optimum sucrose production

The first hurdle we encountered while modelling cyanobacteria was simulating salt stress. Literature says simulating something like salt stress alters a lot of reactions in the organism and not all of them are straight forward or even known, so we had to make a few assumptions to make the model work.

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# Team:IISER-Pune-India/Engineering

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Engineering

##### Team IISER-Pune-India @ iGEM 2021

Team IISER-Pune-India is proud to announce that we've bagged a **gold medal** AND nominations for  **Best Manufacturing Project** and  **Best Education** at iGEM 2021.

Close

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* Team
  + [Members](https://2021.igem.org/Team%3AIISER-Pune-India/Team)
  + [Attributions](https://2021.igem.org/Team%3AIISER-Pune-India/Attributions)
  + [Collaborations](https://2021.igem.org/Team%3AIISER-Pune-India/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3AIISER-Pune-India/Partnership)
  + [Sponsors](https://2021.igem.org/Team%3AIISER-Pune-India/Sponsors)
* Project
  + [Description](https://2021.igem.org/Team%3AIISER-Pune-India/Description)
  + [Design](https://2021.igem.org/Team%3AIISER-Pune-India/Design)
  + [Notebook](https://2021.igem.org/Team%3AIISER-Pune-India/TeamNotebook/Team%20Notebook%2011e9906b405543a29361e38a50c81024)
  + [Contributions](https://2021.igem.org/Team%3AIISER-Pune-India/Contribution)
  + [Engineering Success](https://2021.igem.org/Team%3AIISER-Pune-India/Engineering)
  + [Future Implementation](https://2021.igem.org/Team%3AIISER-Pune-India/Implementation)
  + [Safety](https://2021.igem.org/Team%3AIISER-Pune-India/Safety)
* Wet Lab
  + [Experiments](https://2021.igem.org/Team%3AIISER-Pune-India/Experiments)
  + [Constructs](https://2021.igem.org/Team%3AIISER-Pune-India/Constructs)
  + [Measurement](https://2021.igem.org/Team%3AIISER-Pune-India/Measurement)
  + [Lab Notebook](https://2021.igem.org/Team%3AIISER-Pune-India/LabNotebook)
  + [Results](https://2021.igem.org/Team%3AIISER-Pune-India/Results)
  + [Future Work](https://2021.igem.org/Team%3AIISER-Pune-India/FutureWork)
* Parts
  + [Basic](https://2021.igem.org/Team%3AIISER-Pune-India/BasicParts)
  + [Composite](https://2021.igem.org/Team%3AIISER-Pune-India/CompositeParts)
* [Model](https://2021.igem.org/Team%3AIISER-Pune-India/Model)
* Human Practices
  + [Integrated Human Practices](https://2021.igem.org/Team%3AIISER-Pune-India/Human_Practices)
  + [Inclusivity and Accessibility](https://2021.igem.org/Team%3AIISER-Pune-India/Inclusivity)
  + [Public Engagement and Education](https://2021.igem.org/Team%3AIISER-Pune-India/Communication)
* [Judging Form](https://igem.org/2021_Judging_Form?id=3971)
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# Engineering Success

* [Choosing Optimal promoters](#1)
* [gDNA extraction and PCR](#2)
* [E. coli in CoBG11](#3)
* [Sucrose Assay](#4)
* [S. elongatus growth curves](#5)
* [Co-culture modeling](#6)
* [Building the SteadyCom model](#7)
* [Computational - Experimental Integrated Engineering Cycle](#8)

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](#Cycle1_A)
* [Cycle 2](#Cycle2_A)

### 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](#Cycle1_B)
* [Cycle 2](#Cycle2_B)
* [Cycle 3](#Cycle3_B)
* [Cycle 4](#Cycle4_B)
* [Cycle 5](#Cycle5_B)

### 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](#Cycle1_C)
* [Cycle 2](#Cycle2_C)
* [Cycle 3](#Cycle3_C)

### 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](https://2021.igem.org/Team%3AIISER-Pune-India/Results). |

### References

1. 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](#Cycle1_D)
* [Cycle 2](#Cycle2_D)
* [Cycle 3](#Cycle3_D)

### 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

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

# *S. elongatus* growth curves

---

* [Cycle 1](#Cycle1_E)
* [Cycle 2](#Cycle2_E)
* [Cycle 3](#Cycle3_E)
* [Cycle 4](#Cycle4_E)
* [Cycle 5](#Cycle5_E)

### 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 Tdouble=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

1. 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.
2. 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.

br>

# Co-culture modeling

---

* [Cycle 1](#Cycle1_F)

### 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](#Cycle1_G)
* [Cycle 2](#Cycle2_G)

### 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](#Cycle1_I)
* [Cycle 2](#Cycle2_I)
* [Cycle 3](#Cycle3_I)

### 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. |

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# Team:IISER-Pune-India/Experiments

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Experiments

##### Team IISER-Pune-India @ iGEM 2021

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# Experiments

* [Glycerol stocks of KJK01](#e1)
* [Genomic DNA extraction](#e2)
* [Confirmation of butanol producing genes](#e3)
* [Gradient PCR](#e4)
* [NMR confirmation of butanol production](#e5)
* [Making KJK01 cells electrocompetent](#e6)
* [Transformation of KJK01 with pCSCX plasmid](#e7)
* [Glycerol stocks of pCSCX-KJK01](#e8)
* [MIC Assay for KJK01](#e9)
* [Growth curve in LB](#e10)
* [Sucrose Assay](#e11)

* [Preparation of BG-11](#c1)
* [Revival of *S. elongatus* strains from plates](#c2)
* [Making DMSO and Glycerol Stocks](#c3)
* [Revival of *S. elongatus* strains from glycerol or DMSO stocks](#c4)
* [Growth Curves of *S. elongatus* under salt stress](#c5)
* [Sucrose assay](#c6)
* [References](#c7)

* [Transformation of MG1655 with pKD4](#cc1)
* [Preparation of CoBG-11](#cc2)
* [Growth curve of KJK01 and pCSCX-KJK01 in BG-11 + sucrose](#cc3)
* [Sucrose assay](#cc4)
* [References](#cc5)

* Overview
* *E. coli*
* *S. elongatus*
* Co-culture

Our Wet Lab work was geared towards observing the production of our final product from a co-culture of *E. coli* and *S. elongatus* UTEX 2973. To that end, we broke it down into four steps:

1. Engineering our cyanobacteria to secrete sucrose under salt stress;
2. Engineering our *E. coli* to produce our final product (butanol or succinate);
3. Engineering the *E. coli* to consume sucrose;
4. Growing a co-culture of the two

Then came the devastating second wave of COVID-19, cutting our lab time down from seven months to two. We were incredibly fortunate, in that situation, to benefit from the help of Dr Himadri Pakrasi and Dr Shams Yazdani. Dr Pakrasi's lab provided us with sucrose-secreting strains they had previously engineered - UTEX 2973-*cscB*, 2973-cscB-*spp* and 2973-cscB-*spp*-*spp*. *S. elongatus*'s freshwater strains naturally produce sucrose as an osmoprotectant, and the presence of *cscB* (coding for sucrose permease) leads to that sucrose being secreted into the growth medium[[1]](#oref1). The genes *spp* and *spp* upregulate the sucrose production pathway. This was a huge relief for us, as UTEX 2973 is not naturally transformable. Genes have to be introduced via triparental mating (a 9-day-long protocol involving conjugation) and incorporated into the genome via a homologous recombination strategy, such as CRISPR. Dr Yazdani's lab lent us his *E. coli* KJK01 strain - an IPTG-inducible butanol-producing derivative of MG1655.[[2]](#oref2) The genes controlling the butanol-producing pathway have been markerlessly integrated into the genome, making it easy to work with for the purposes of our co-culture.

Our immediate work in the lab, then, was to test out the strains we'd received and to set up a co-culture. We used NMR to assay for the presence of butanol in the LB medium of the KJK01. Following confirmation of butanol production, we made the KJK01 cells electrocompetent and transformed them with pCSCX - a plasmid containing genes for sucrose uptake and digestion.[[3]](#oref3) pCSCX is marked with Kanamycin and Ampicillin, the former of which is also shared by UTEX-2973, making it useful for ensuring the purity of our co-culture. For the cyanobacteria, we used Megazyme's kit for a colorimetric sucrose assay to establish a standard curve of fluorescence versus sucrose concentration, and used it to quantify the sucrose concentration in supernatant aliquots taken at regular intervals.[[4]](#oref4) The same assay and standard curve were used to quantify sucrose consumption in KJK01 grown in Co-BG11 with sucrose. Co-BG11 is a variant of the standard BG11 medium for cyanobacteria, and is free of any other carbon source; this variant is optimised for co-cultures with *E. coli*.[[5]](#oref5)

Reviving cyanobacteria was a difficult task and involved setting up lights in our incubator, acquiring a lux meter and making many, many mistakes as it is a relatively new model organism. We maintained a record of everything we learn from our mentors at the Wangikar lab and are publishing it as a contribution to future teams working on cyanobacteria.

We intend in future work to re-engineer the cyanobacteria to overexpress the sucrose production pathway and the sucrose permease gene under strong native promoters, and to choose a promoter that responds well to high CO2 conditions for our final application. This is principally on the advice of Dr Shireesh Srivastava, who has observed in the course of his work that native promoters often lead to higher and more consistent levels of expression with less detriment to the host. We further intend to find optimal conditions - light, carbon dioxide, reductants to remove oxygen evolved by cyanobacteria, temperature and so forth - allow for a stable and productive co-culture.

For our E. coli KJK01 strain, we plan to characterize the bidirectional promoter present in our pcscX plasmid. This will help us understand the flux through the csc gene products and their importance for our strain. We further plan to increase the butanol tolerance of our strain based on studies by Reyes et al.[[6]](#oref6) and Boyarskiy et al.[[7]](#oref7) For further information on this check out our [proposed implementation](#Implementation.html). We will also perform our growth curves with this modified strain. We also plan to incorporate our drylab results into our wetlab by knocking out and overexpressing the genes suggested by OptKnock and FSEOF respectively. We further plan to optimize our co culture using the results from our modified strains.

# References

---

1. Lin, P. C., Zhang, F., & Pakrasi, H. B. (2020). Enhanced production of sucrose in the fast-growing cyanobacterium *Synechococcus elongatus* UTEX 2973. Scientific reports, 10(1), 1-8.
2. Abdelaal, A. S., Jawed, K., & Yazdani, S. S. (2019). CRISPR/Cas9-mediated engineering of Escherichia coli for n-butanol production from xylose in defined medium. Journal of Industrial Microbiology and Biotechnology, 46(7), 965-975.
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. [Megazyme's sucrose assay kit](https://www.megazyme.com/sucrose-d-glucose-assay-kit)
5. 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.
6. Reyes, L. H., Almario, M. P., & Kao, K. C. (2011). Genomic library screens for genes involved in n-butanol tolerance in Escherichia coli. PloS one, 6(3), e17678.
7. Sergey Boyarskiy, Stephanie Davis LÃ³pez, Niwen Kong, Danielle Tullman-Ercek,Transcriptional feedback regulation of efflux protein expression for increased tolerance to and production of n-butanol,Metabolic Engineering,Volume 33,2016,Pages 130-137,ISSN 1096-7176

The experiments below are presented in the order in which we performed them.

## Glycerol stocks of KJK01

### Objective

To prepare glycerol stocks of the *E. coli* KJK01 strain.

### Working Principle[[1]](#eref1)

Bacterial cultures in the form of agar plates or liquid cultures are not suitable for long term storage at low temperatures such as 4°. Long term storage of bacteria is best at temperatures around -80°. However, agar plates and liquid cultures cannot be stored at such temperatures, as the water crystallizes in and around the bacterial cells, rupturing and killing them. Glycerol, an anti-freezing agent, is added to the media to store samples at such temperatures.

### Key Reagents/Apparatus

* Autoclaved 1X LB
* Autoclaved 50% glycerol

* Cryovials

### Protocol

Can be found [here](https://www.protocols.io/view/glycerol-stock-preparation-byyppxvn)

![Flash freezing the prepared KJK01 stocks](https://static.igem.org/mediawiki/2021/9/9c/T--IISER-Pune-India--WetlabPhotos--Glycerol_stocks_3.jpeg)

---

## Genomic DNA extraction

### Objective

To extract the genomic DNA of *E. coli* KJK01 to verify the presence of the *hbd-crt* butanol production pathway genes.

### Working principle[[2]](#eref2)

To extract the genomic DNA from the *E. coli* cells, the cell membrane is first lysed using SDS to release cellular contents. Proteins and RNA from the cells are destroyed using Proteinase K and RNase A respectively. This leaves behind only DNA, which is precipitated in isopropanol.

Once the gDNA has been extracted, it can be run on an agarose gel to verify its presence.

### Key Reagents/Apparatus

* Sodium dodecyl sulfate (SDS)
* Proteinase K
* RNase A
* Isopropanol
* Ethanol
* Phenol-chloroform
* Double distilled water (MilliQ water)
* Sodium Acetate
* TAE buffer
* Ethidium Bromide
* DNA Ladder

* Centrifuge
* Thermocycler
* Gel Electrophoresis Machine
* Gel imager
* NanoDrop

### Protocol

Can be found [here](http://dx.doi.org/10.17504/protocols.io.bxeupjew)

---

## Confirmation of butanol producing genes

### Objective

To confirm the presence of butanol producing genes in KJK01 strain using PCR and gel electrophoresis.

### Working principle[[3]](#eref3)

The butanol producing ability of KJK01 is due to the presence of the cassette of genes inserted into the genome of the strain. 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.

Primers against the *hbd-crt* genes (sequence provided by Dr Yazdani) were used to check for the presence of these 2 genes from the genomic DNA of KJK01. An amplicon of length of ~750bp from the PCR would verify the presence of *hbd-crt* genes.

### Key Reagents/Apparatus

* Pfu polymerase
* Pfu buffer
* dNTP mix
* Primers
* Agarose
* 1X TAE
* EtBr solution

* Thermocycler
* Gel electrophoresis apparatus
* Gel dock

### Protocol

Can be found [here](https://www.protocols.io/view/pcr-and-gel-electropheresis-bxempjc6)

---

## Gradient PCR

### Objective

To perform a gradient PCR to find the best annealing temperature as well as the best amount of DMSO to add to obtain proper amplification of the gene of interest.

### Working principle[[4]](#eref4)

Thermocyclers used for PCR reactions can maintain different tubes at different temperatures in the same run. This is exploited to vary the annealing temperature and observe which of the set temperatures is best suited for annealing and thus gives best amplification. This allows for a single PCR run to give us results for multiple annealing temperatures.

DMSO prevents the renaturation of single strands of DNA. Thus, it helps in amplification of the gene of interest.[[4]](#eref4) An optimum amount can be found by adding different amounts to tubes in gradient PCR.

### Key Reagents/Apparatus

* Pfu polymerase
* Pfu buffer
* dNTP mix
* Primers
* Agarose
* 1X TAE
* EtBr solution
* DMSO

* Thermocycler
* Gel electrophoresis apparatus
* Gel dock

### Protocol

Can be found [here](https://dx.doi.org/10.17504/protocols.io.by6kpzcw)

---

## NMR confirmation of butanol production

### Objective

To confirm production of butanol by *E. coli* KJK01 using NMR spectroscopy

### Working principle [[5]](#eref5)

Nuclear magnetic resonance (NMR) spectroscopy, is a spectroscopic technique to observe local magnetic fields around atomic nuclei based on their absorption of electromagnetic radiation in the radio-frequency region 4 to 900 MHz. The principle behind NMR is that many nuclei have a spin and all nuclei are electrically charged. If an external magnetic field is applied, energy transfer is possible between the ground state to a higher energy level (generally a single energy gap). The energy transfer takes place at a wavelength that corresponds to appropriate radio frequencies and when the spin returns to its base level, energy is emitted at the same frequency. The signal that matches this transfer is measured and processed in order to yield an NMR spectrum for the nucleus concerned. Fields are highly characteristic of individual compounds. NMR also provides detailed information about the structure, dynamics, reaction state, and chemical environment of molecules.

We used proton NMR for the detection of butanol in the supernatant of our *E. coli* KJK01 cultures, alongside PCR confirmation of butanol producing genes.

### Key Reagents/Apparatus

* Supernatants of IPTG induced *E. coli* KJK01 2° culture

### Protocol

1. Inoculate 1° culture of KJK01 in 10 mL 1X LB and leave it overnight in an incubator at 37°C and 180 rpm.
2. The next morning, inoculate 2° cultures of KJK01 in 1X LB and 1X TB and place in the incubator at 37°C and 180 rpm until it hits 0.6 OD600.

* We inoculated 6 2° cultures (3 LB and 3 TB) to confirm if KJK01 grew better in LB or TB (The secondary culture was originally cultured in TB expecting better growth[[3]](#eref3))

3. At OD 0.6, induce cultures with appropriate concentrations of IPTG and leave it in the incubator overnight at 37°C and 180 rpm.

* The 6 cultures are:

+ 100 µL 1° culture + 10 mL 1X LB + 0 IPTG
+ 100 µL 1° culture + 10 mL 1X LB + 1 µL (0.1 mM) IPTG
+ 100 µL 1° culture + 10 mL 1X LB + 2 µL (0.2 mM) IPTG
+ 100 µL 1° culture + 10 mL 1X TB + 0 IPTG
+ 100 µL 1° culture + 10 mL 1X TB + 1 µL (0.1 mM) IPTG
+ 100 µL 1° culture + 10 mL 1X TB + 2 µL (0.2 mM) IPTG

4. The next morning, draw 1 mL sample from each culture and centrifuge at 4° and 5000 rpm for 15 minutes.
5. Decant the solution and submit supernatant for NMR analysis.

Soumya Sahoo, a PhD student at the NMR Lab of IISER Pune helped us with the NMR analysis of our samples.

---

## Making KJK01 cells electrocompetent

### Objective

To prepare KJK01 electrocompetent cells

### Working principle

To transform any cells, they need to be competent. Only competent cells can take up DNA. Competent cells can be chemically or electrically stimulated to open up the cell pores and take up DNA. The preparation of cells for chemical or electrical transformation differs in protocol, and the cells competent for the same are called chemically competent and electrocompetent cells, respectively.

Electrocompetent cells can be transformed using an electrical pulse. When electro-competent cells are pulsed, the pulse will rearrange the lipid bilayer to form a gap for the intake of the plasmid [[7]](#eref7).

The preparation of electrocompetent cells involves washing the grown cells serially using water and glycerol multiple times. These washes are important to remove all the salts surrounding the cells. The salts would otherwise cause sparking during the electrical pulse, damaging the apparatus and the cell wall. Competent cells are finally mixed in dilute glycerol solution, flash frozen and stored at -80°.

### Key Reagents/Apparatus

* Autoclaved 2X LB
* Autoclaved 10% glycerol

* Centrifuge
* Liq. Nitrogen

### Protocol

Can be found [here](https://www.protocols.io/view/making-electro-competent-cells-bxxhppj6)

---

## Transformation of KJK01 with pCSCX plasmid

### Objective

To transform *E. coli* KJK01 with plasmid pCSCX

### Working principle [[7]](#eref7)

Transformation via electroporation is a physical transformation method that uses an electrical pulse to create temporary pores in cell membranes through which substances like nucleic acids can pass into cells. Host cells and nucleic acids are suspended in a conductive solution, and an electrical circuit is closed around the mixture. An electrical pulse at an optimized voltage lasting a few microseconds to a millisecond is discharged through the cell suspension, which disturbs the phospholipid bilayer of the membrane and results in the formation of temporary pores. The electric potential across the cell membrane simultaneously rises to allow charged molecules like DNA to be driven across the membrane through the pores. This method can transform a large number of cells in a short period of time as it is easy and rapid.

The pCSCX gene contains *cscABK* genes from *E. coli* W that help in transport and metabolism of sucrose. *E. coli* strains that have been transformed with this plasmid have shown to be able to survive with sucrose as their sole carbon source[[8]](https://2021.igem.org/Team%3AIISER-Pune-India/eref8). We hope to replicate these results.

### Key Reagents/Apparatus

* Electrocompetent cells
* Plasmid DNA
* 2X LB
* MilliQ water

* BioRad Pulser

### Protocol

Can be found [here](https://www.protocols.io/view/transformation-of-e-coli-with-plasmid-byz3px8n)

![Best 5 over-expression targets in one graph](https://static.igem.org/mediawiki/2021/6/61/T--IISER-Pune-India--WetlabPhotos--Tranformation_1.jpeg)

![Next best 5 over-expression targets in one graph](https://static.igem.org/mediawiki/2021/6/63/T--IISER-Pune-India--WetlabPhotos--Transformation_2.jpeg)

![Third best 5 over-expression targets in one graph](https://static.igem.org/mediawiki/2021/2/22/T--IISER-Pune-India--WetlabPhotos--Transformation_3.jpeg)

![Fourth best 5 over-expression targets in one graph](https://static.igem.org/mediawiki/2021/d/dd/T--IISER-Pune-India--WetlabPhotos--Tranformation_4.jpeg)

Previous

Next

---

## Glycerol stocks of pCSCX-KJK01

### Objective

To prepare glycerol stocks of the *E. coli* KJK01 strain transformed with the *cscABK* gene containing pCSCX plasmid.

### Working principle

Bacterial cultures in the form of agar plates or liquid cultures are not suitable for long term storage at low temperatures such as 4°. Long term storage of bacteria is best at temperatures around -80°. However, agar plates and liquid cultures cannot be stored at such temperatures, as the water crystallizes in and around the bacterial cells, rupturing and killing them. Glycerol, an anti-freezing agent, is added to the media to store samples at such temperatures.[[1]](#eref1)

### Key Reagents/Apparatus

* Autoclaved 1X LB
* Autoclaved 50% glycerol

* Cryovials

### Protocol

Can be found [here](https://www.protocols.io/view/glycerol-stock-preparation-byyppxvn)

![Best 5 over-expression targets in one graph](https://static.igem.org/mediawiki/2021/f/f3/T--IISER-Pune-India--WetlabPhotos--Transformed_stocks_1.jpeg)

![Next best 5 over-expression targets in one graph](https://static.igem.org/mediawiki/2021/9/9d/T--IISER-Pune-India--WetlabPhotos--Transformed_stocks_2.jpeg)

Previous

Next

---

## MIC Assay for KJK01

### Objective

To determine the minimum inhibitory concentration (MIC) of butanol and ethanol for  *E. coli* KJK01 strain.

### Working principle

Minimum inhibitory concentration (MIC) is defined as the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation. [[9]](#eref9) Determination of MIC involves obtaining a growth curve of the strain at varying concentrations of the antimicrobial and identifying the least concentration at which growth is inhibited.

This experiment will help us calculate the optimum extraction frequency of products from the bioreactor medium in order to maximise yield from the cells.

### Key Reagents/Apparatus

* MG1655 2° culture (Control strain)
* KJK01 2° cultures (uninduced and induced with IPTG)
* 1X LB
* MilliQ

* 96-well plates

### Protocol

Can be found [here](https://www.protocols.io/view/minimum-inhibitory-concentration-of-butanol-for-e-byz8px9w)

![Best 5 over-expression targets in one graph](https://static.igem.org/mediawiki/2021/2/29/T--IISER-Pune-India--WetlabPhotos--MIC_1.jpeg)

![Next best 5 over-expression targets in one graph](https://static.igem.org/mediawiki/2021/d/d4/T--IISER-Pune-India--WetlabPhotos--MIC_2.jpeg)

![Third best 5 over-expression targets in one graph](https://static.igem.org/mediawiki/2021/0/05/T--IISER-Pune-India--WetlabPhotos--MIC_3.jpeg)

![Fourth best 5 over-expression targets in one graph](https://static.igem.org/mediawiki/2021/6/6e/T--IISER-Pune-India--WetlabPhotos--MIC_4.jpeg)

Previous

Next

---

## Growth Curve in LB

### Objective

To obtain growth curves of KJK01 and pCSCX-KJK01 in LB and to characterize their growth

### Working principle[[10]](#eref10)

When bacteria are inoculated into a liquid medium and the cell population is counted at intervals, it is possible to plot a typical bacterial growth curve that shows the growth of cells over time. It shows four distinct phases of growth.

* Lag phase: Slow growth or lack of growth due to physiological adaptation of cells to culture conditions or dilution of exoenzymes due to initial low cell densities.
* Log or exponential phase: Optimal growth rates, during which cell numbers double at discrete time intervals known as the mean generation time.
* Stationary phase: Growth (cell division) and death of cells counterbalance each other resulting in no net increase in cell numbers. The reduced growth rate is usually due to a lack of nutrients and/or a buildup of toxic waste constituents.
* Decline or death phase: Death rate exceeds growth rate resulting in a net loss of viable cells.

This is one of the simplest methods used to analyze trends in growth because it uses a spectrophotometer to track changes in the optical density (OD) over time. In other words, as the number of cells in a sample increases, the transmission of light through the sample will decrease.

### Key Reagents/Apparatus

* 2° cultures of KJK01 and pCSCX-KJK01 in LB

* Photospectrometer
* Quartz/plastic (disposable) cuvette

### Protocol

Can be found [here](https://www.protocols.io/view/growth-rate-determination-of-e-coli-bxa2pige)

---

## Sucrose Assay

### Objective

To measure the consumption of sucrose by *E. coli* KJK01 and pCSCX-KJK01

Same as for *S. elongatus*, except we look for decrease in sucrose concentration as opposed to increase. Link to sucrose assay for *S. elongatus* can be found [here](#c6).

### Working Principle

The sucrose assay is a colorimetric assay[[11]](#eref11) to quantify sucrose concentration in cell supernatants. Supernatant samples from the cultures are taken at regular frequencies. A decreasing trend in the absorbance of successive samples after treatment is indicative of decreasing sucrose concentration, i.e, net sucrose consumption has been taking place.

* Distilled water
* D-Glucose standard solution (5 mL, 1.0 mg/mL) in 0.2% (w/v) benzoic acid
* GOPOD Reagent Enzymes. Glucose oxidase plus peroxidase and 4-aminoantipyrine. Freeze-dried powder
* GOPOD Reagent Buffer. p-hydroxybenzoic acid and sodium azide (0.095% w/v)
* Î²-Fructosidase (invertase) solution
* Acetate buffer

* 96 well plate
* 1 mL Eppendorf tubes
* Spectrophotometer set at 510 nm
* Vortex mixer
* Thermostated water bath

# Reference

---

1. <https://www.addgene.org/protocols/create-glycerol-stock/>
2. Surzycki, Stefan (2000). Basic Techniques in Molecular Biology || Preparation of Genomic DNA from Bacteria. , 10.1007/978-3-642-56968-5(Chapter 4), 79-100. doi:10.1007/978-3-642-56968-5\_4
3. Abdelaal, A. S., Jawed, K., & Yazdani, S. S. (2019). CRISPR/Cas9-mediated engineering of Escherichia coli for n-butanol production from xylose in defined medium. Journal of Industrial Microbiology and Biotechnology, 46(7), 965-975.
4. Gopalakrishnan, Guruprasad. (2012). Re: What exactly is the function of DMSO in PCR? . Retrieved from: [research gate](https://www.researchgate.net/post/What_exactly_is_the_function_of_DMSO_in_PCR/500ad731e4f076bb14000011/citation/download)
5. <https://www.sciencedirect.com/topics/materials-science/nuclear-magnetic-resonance-spectroscopy>
6. <https://www.goldbio.com/articles/article/Introduction-to-Competent-Cells>
7. <https://www.thermofisher.com/in/en/home/references/gibco-cell-culture-basics/transfection-basics/transfection-methods/electroporation.html>
8. 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>
9. Andrews J. M. (2001). Determination of minimum inhibitory concentrations. The Journal of antimicrobial chemotherapy, 48 Suppl 1, 5-16. <https://doi.org/10.1093/jac/48.suppl_1.5>
10. Microbial Growth. (2021, January 4). Oregon State University https://bio.libretexts.org/@go/page/10667.

The experiments below are presented in the order in which we performed them.

## Preparation of BG-11

### Objective

To prepare BG-11 media for culturing of cyanobacteria

### Working principle

BG-11 media is a mix of macronutrients and micronutrients without essentially a consumable carbon source. Since this media is used for culturing cyanobacteria which are capable of photosynthesizing, a consumable carbon source is not required.

BG-11 mix has: sodium nitrate, dipotassium hydrogen phosphate, magnesium sulphate heptahydrate, calcium chloride dihydrate, citric acid, sodium carbonate, sodium EDTA, ferric ammonium citrate and micronutrients (trace metals) mix.

Adjusting the pH of the media according to the optimal growth pH of the cyanobacteria is crucial for BG-11. It also so happens that post autoclaving the pH might change. In such cases it is always good to check the pH of an aliquot of sterile media before using.

### Key Reagents/Apparatus

* BG-11 powder
* Trace metal mix
* 1N HCl
* 1N NaOH

### Protocol

* For liquid media can be found [here](https://dx.doi.org/10.17504/protocols.io.by6qpzdw)
* For Media agar can be found [here](https://dx.doi.org/10.17504/protocols.io.by6rpzd6)

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# Team:IISER-Pune-India/Results

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Results

##### Team IISER-Pune-India @ iGEM 2021

Team IISER-Pune-India is proud to announce that we've bagged a **gold medal** AND nominations for  **Best Manufacturing Project** and  **Best Education** at iGEM 2021.

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# Results

* [PCR and gel electrophoresis for confirmation of butanol producing genes](#e1)
* [NMR confirmation of butanol production](#e2)
* [Transformation of KJK01 with pCSCX plasmid](#e3)
* [Growth curve assay for KJK01 and pCSCX-KJK01](#e4)
* [MIC assay of butanol and ethanol for KJK01](#e5)
* [Sucrose assay for KJK01 in LB](#e6)
* [References](#e7)

* [Growth Curve - Ambient carbon dioxide](#c1)
* [Sucrose Assay](#c2)
* [Growth Curve - High carbon dioxide](#c3)
* [References](#c4)

* [Growth curve assay for KJK01 and pCSCX-KJK01 in BG-11 + sucrose](#cc1)
* [Transformation of KJK01 with pKD4 plasmid](#cc2)
* [Growth curve assay for KJK01 and pCSCX-KJK01 in CoBG-11 + sucrose](#cc3)
* [Sucrose assay](#cc4)
* [References](#cc5)

* *E. coli*
* *S. elongatus*
* Co-culture

# 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]](#eref1). 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.

![](https://static.igem.org/mediawiki/2021/6/6e/T--IISER-Pune-India--Wetlab_Graphs--E_coli--Gels--gDNA.png)

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.

![](https://static.igem.org/mediawiki/2021/d/d6/T--IISER-Pune-India--Wetlab_Graphs--E_coli--Gels--PCR_amp.png)

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.

read more

PCR with gDNA extract 2:

No bands appeared on imaging the PCR product.

## Gradient PCR

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.

![](https://static.igem.org/mediawiki/2021/1/1e/T--IISER-Pune-India--Wetlab_Graphs--E_coli--Gels--Gradient_PCR.png)

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]](#eref1) 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

![](https://static.igem.org/mediawiki/2021/5/58/T--IISER-Pune-India--Wetlab_Graphs--E_coli--NMR--NMR_LBoverlay_1.png)

NMR spectrum for LB 2&#176 cultures

![](https://static.igem.org/mediawiki/2021/2/2e/T--IISER-Pune-India--Wetlab_Graphs--E_coli--NMR--NMR_LBoverlay_2.png)

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

![](https://static.igem.org/mediawiki/2021/6/6e/T--IISER-Pune-India--Wetlab_Graphs--E_coli--NMR--NMR_TBoverlay_1.png)

NMR spectrum for TB 2&#176 cultures

![](https://static.igem.org/mediawiki/2021/a/a7/T--IISER-Pune-India--Wetlab_Graphs--E_coli--NMR--NMR_TBoverlay_2.png)

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]](#eref3) 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.

![](https://static.igem.org/mediawiki/2021/b/b9/T--IISER-Pune-India--Wetlab_Graphs--E_coli--Plates--Tranformation_plate_1.jpeg)

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:

![](https://static.igem.org/mediawiki/2021/d/df/T--IISER-Pune-India--Wetlab_Graphs--E_coli--Growth--LB_Growthcurve_KJK01.png)

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

![](https://static.igem.org/mediawiki/2021/b/b7/T--IISER-Pune-India--Wetlab_Graphs--E_coli--Growth--LB_Growthcurve_KJK01_transformed.png)

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]](#eref2)
+ 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]](#eref4),[[5]](#eref5) Since we have not made any modifications in KJK01 to increase it’s butanol tolerance, we expect similar results in this strain.

#### Results

![Best 5 over-expression targets in one graph](https://static.igem.org/mediawiki/2021/b/b9/T--IISER-Pune-India--Wetlab_Graphs--E_coli--MIC--Plate_1_MG1655_b.png)

![Next best 5 over-expression targets in one graph](https://static.igem.org/mediawiki/2021/4/48/T--IISER-Pune-India--Wetlab_Graphs--E_coli--MIC--Plate_1_KJK01_b.png)

![Third best 5 over-expression targets in one graph](https://static.igem.org/mediawiki/2021/d/d1/T--IISER-Pune-India--Wetlab_Graphs--E_coli--MIC--Plate_1_KJK01_ind_b.png)

![Fourth best 5 over-expression targets in one graph](https://static.igem.org/mediawiki/2021/3/3e/T--IISER-Pune-India--Wetlab_Graphs--E_coli--MIC--Plate_1_MG1655_e.png)

![Next best 5 over-expression targets in one graph](https://static.igem.org/mediawiki/2021/a/a4/T--IISER-Pune-India--Wetlab_Graphs--E_coli--MIC--Plate_1_KJK01_e.png)

![Next best 5 over-expression targets in one graph](https://static.igem.org/mediawiki/2021/6/65/T--IISER-Pune-India--Wetlab_Graphs--E_coli--MIC--Plate_1_KJK01_ind_e.png)

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MIC Graphs for plate 1

![Best 5 over-expression targets in one graph](https://static.igem.org/mediawiki/2021/d/d5/T--IISER-Pune-India--Wetlab_Graphs--E_coli--MIC--Plate_2_MG1655_b.png)

![Next best 5 over-expression targets in one graph](https://static.igem.org/mediawiki/2021/6/6b/T--IISER-Pune-India--Wetlab_Graphs--E_coli--MIC--Plate_2_KJK01_b.png)

![Third best 5 over-expression targets in one graph](https://static.igem.org/mediawiki/2021/d/dd/T--IISER-Pune-India--Wetlab_Graphs--E_coli--MIC--Plate_2_KJK01_ind_b.png)

![Fourth best 5 over-expression targets in one graph](https://static.igem.org/mediawiki/2021/0/00/T--IISER-Pune-India--Wetlab_Graphs--E_coli--MIC--Plate_2_MG1655_e.png)

![Next best 5 over-expression targets in one graph](https://static.igem.org/mediawiki/2021/4/45/T--IISER-Pune-India--Wetlab_Graphs--E_coli--MIC--Plate_2_KJK01_e.png)

![Next best 5 over-expression targets in one graph](https://static.igem.org/mediawiki/2021/7/78/T--IISER-Pune-India--Wetlab_Graphs--E_coli--MIC--Plate_2_KJK01_ind_e.png)

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MIC Graphs for plate 2

![Best 5 over-expression targets in one graph](https://static.igem.org/mediawiki/2021/0/0f/T--IISER-Pune-India--Wetlab_Graphs--E_coli--MIC--Plate_3_MG1655_b.png)

![Next best 5 over-expression targets in one graph](https://static.igem.org/mediawiki/2021/e/ef/T--IISER-Pune-India--Wetlab_Graphs--E_coli--MIC--Plate_3_KJK01_b.png)

![Third best 5 over-expression targets in one graph](https://static.igem.org/mediawiki/2021/1/19/T--IISER-Pune-India--Wetlab_Graphs--E_coli--MIC--Plate_3_KJK01_ind_b.png)

![Fourth best 5 over-expression targets in one graph](https://static.igem.org/mediawiki/2021/f/fa/T--IISER-Pune-India--Wetlab_Graphs--E_coli--MIC--Plate_3_MG1655_e.png)

![Next best 5 over-expression targets in one graph](https://static.igem.org/mediawiki/2021/d/da/T--IISER-Pune-India--Wetlab_Graphs--E_coli--MIC--Plate_3_KJK01_e.png)

![Next best 5 over-expression targets in one graph](https://static.igem.org/mediawiki/2021/d/d8/T--IISER-Pune-India--Wetlab_Graphs--E_coli--MIC--Plate_3_KJK01_ind_e.png)

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MIC Graphs for plate 3

Strain

Compound

MIC (mg/mL)

Comments

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

![](https://static.igem.org/mediawiki/2021/4/4f/T--IISER-Pune-India--WetlabPhotos--SucroseAssay_ecoli.png)

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

![](https://static.igem.org/mediawiki/2021/c/cf/T--IISER-Pune-India--Wetlab_Graphs--E_coli--Sucroseassay_ecoli_result.png)

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

![](https://static.igem.org/mediawiki/2021/9/9d/T--IISER-Pune-India--pngs--Mascot_ecoli.png)

---

### 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]](#cref1). 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

![](https://static.igem.org/mediawiki/2021/4/4e/T--IISER-Pune-India--Wetlab_Graphs--Cyanobacteria--Cyano_growthcurve1_WT_logisticinv.png)

![](https://static.igem.org/mediawiki/2021/7/76/T--IISER-Pune-India--pngs--Mascot_cyano.png)

* *S. elongatus* UTEX 2973-*cscB*

![](https://static.igem.org/mediawiki/2021/2/21/T--IISER-Pune-India--Wetlab_Graphs--Cyanobacteria--Cyano_growthcurve1_cscB_logisticinv.png)

![](https://static.igem.org/mediawiki/2021/9/9d/T--IISER-Pune-India--pngs--Mascot_ecoli.png)

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]](#cref2)

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]](#cref3) 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]](#cref4) 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.

![](https://static.igem.org/mediawiki/2021/5/59/T--IISER-Pune-India--Wetlab_Graphs--Cyanobacteria--SucroseAssay_1.png)

![](https://static.igem.org/mediawiki/2021/7/76/T--IISER-Pune-India--pngs--Mascot_cyano.png)

# 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]](#cref5). 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)

![](https://static.igem.org/mediawiki/2021/4/49/T--IISER-Pune-India--Wetlab_Graphs--Cyanobacteria--Cyano_growthcurve_HC_WT_loginv.png)

![](https://static.igem.org/mediawiki/2021/7/76/T--IISER-Pune-India--pngs--Mascot_cyano.png)

* *cscB*(IPTG+)

![](https://static.igem.org/mediawiki/2021/1/15/T--IISER-Pune-India--Wetlab_Graphs--Cyanobacteria--Cyano_growthcurve_cscb_IPTG%2B.png)

![](https://static.igem.org/mediawiki/2021/7/76/T--IISER-Pune-India--pngs--Mascot_cyano.png)

* *cscB*(IPTG-)

![](https://static.igem.org/mediawiki/2021/9/98/T--IISER-Pune-India--Wetlab_Graphs--Cyanobacteria--Cyano_growthcurve_cscb_IPTG-.png)

![](https://static.igem.org/mediawiki/2021/9/9d/T--IISER-Pune-India--pngs--Mascot_ecoli.png)

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]](#ccref1) 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]](#ccref2). 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:

![](https://static.igem.org/mediawiki/2021/b/b4/T--IISER-Pune-India--Wetlab_Graphs--Co_Culture--Growth_Curve_BG11--BG11_Sucrose_GrowthCurve1_KJK01_1509.png)

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

![](https://static.igem.org/mediawiki/2021/c/ca/T--IISER-Pune-India--Wetlab_Graphs--Co_Culture--Growth_Curve_BG11--BG11_Sucrose_GrowthCurve1_KJK01Transformed_induced_1509.png)

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

![](https://static.igem.org/mediawiki/2021/8/82/T--IISER-Pune-India--Wetlab_Graphs--Co_Culture--Growth_Curve_BG11--BG11_Sucrose_GrowthCurve1_KJK01_Transformed_1509.png)

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

![](https://static.igem.org/mediawiki/2021/9/9d/T--IISER-Pune-India--pngs--Mascot_ecoli.png)

* 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.

![](https://static.igem.org/mediawiki/2021/4/41/T--IISER-Pune-India--Wetlab_Graphs--Co_Culture--Growth_Curve_BG11--BG11_Sucrose_GrowthCurve1_KJK01_1709.png)

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

![](https://static.igem.org/mediawiki/2021/a/ae/T--IISER-Pune-India--Wetlab_Graphs--Co_Culture--Growth_Curve_BG11--BG11_Sucrose_GrowthCurve1_KJK01_transformed_induced_1709.png)

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

![](https://static.igem.org/mediawiki/2021/f/f0/T--IISER-Pune-India--Wetlab_Graphs--Co_Culture--Growth_Curve_BG11--BG11_Sucrose_GrowthCurve1_KJK01_Transformed_1_1709.png)

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

![](https://static.igem.org/mediawiki/2021/9/9d/T--IISER-Pune-India--pngs--Mascot_ecoli.png)

* 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]](#ccref3)

#### 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]](#ccref4).

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]](#ccref5) 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]](#cc5) 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.

![](https://static.igem.org/mediawiki/2021/3/3a/T--IISER-Pune-India--Wetlab_Graphs--Co_Culture--Growth_Curve_BG11--CoBG11_Sucrose_GrowthCurve1_KJK01_.png)

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

![](https://static.igem.org/mediawiki/2021/9/92/T--IISER-Pune-India--Wetlab_Graphs--Co_Culture--Growth_Curve_BG11--CoBG11_Sucrose_GrowthCurve1_KJK01_transformed.png)

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

![](https://static.igem.org/mediawiki/2021/0/01/T--IISER-Pune-India--Wetlab_Graphs--Co_Culture--Growth_Curve_BG11--CoBG11_Sucrose_GrowthCurve1_KJK01_transformed_induced.png)

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

![](https://static.igem.org/mediawiki/2021/7/76/T--IISER-Pune-India--pngs--Mascot_cyano.png)

#### 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]](#ccref5) 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]](#ccref2). 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]](#ccref6), 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](assets/Wetlab_Graphs/Raw_data.rar)

---

### 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>
3. 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>
4. Datsenko, K. A., & Wanner, B. L. (2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proceedings of the National Academy of Sciences of the United States of America, 97(12), 6640–6645. <https://doi.org/10.1073/pnas.120163297>
5. Zhang, L., Chen, L., Diao, J. et al. 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 CO2. Biotechnol Biofuels 13, 82 (2020). <https://doi.org/10.1186/s13068-020-01720-0>
6. <https://www.megazyme.com/sucrose-d-glucose-assay-kit>

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