iGEM 2022 — Team shanghaitech-china

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each
day for your project.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/4115/wiki/logowhite.svg)
![](https://static.igem.wiki/teams/4115/wiki/logowhite.svg)

Notebook

| Description | Record |
| --- | --- |
| Surose utilization in *E. coli* | [Wet.1.1 Surose utilization in *E. coli*](https://static.igem.wiki/teams/4115/wiki/wet-1-1-sucrose-utilizing-in-e-coli.pdf) |
| Starvation response in *E. coli* | [Wet.1.2 Starvation response in *E. coli*](https://static.igem.wiki/teams/4115/wiki/wet-1-2-starvation-response-in-e-coli.pdf) |
| Quorum sensing in *E. coli* | [Wet.1.3 Quorum sensing in *E. coli*](https://static.igem.wiki/teams/4115/wiki/wet-1-3-quorum-sensing-in-e-coli.pdf) |
| Culture and Operation of *S. elongatus* | [Wet.2.1 Culture and Operation of S. elongatus.pdf](https://static.igem.wiki/teams/4115/wiki/wet-2-1-culture-and-operation-of-s-elongatus.pdf) |
| Plasmid construction containing CscB *S. elongatus* | [Wet.2.2 Plasmid construction containing CscB.pdf](https://static.igem.wiki/teams/4115/wiki/wet-2-2-plasmid-construction-containing-cscb.pdf) |
| Plasmid construction containing LuxR for *S. elongatus* | [Wet.2.3 Plasmid construction containing LuxR.pdf](https://static.igem.wiki/teams/4115/wiki/wet-2-3-plasmid-construction-containing-luxr.pdf) |
| Plasmid construction related to gene silencing for *S. elongatus*(uncompleted experiment) | [Wet.2.4 Plasmid construction related to gene silencing(uncompleted experiment).pdf](https://static.igem.wiki/teams/4115/wiki/wet-2-4-plasmid-construction-related-to-gene-silencing-uncompleted-experiment.pdf) |
| Culture and Operation of *A. caulinodans* | [Wet.3.1 Culture and Operation of *A. caulinodans*.pdf](https://static.igem.wiki/teams/4115/wiki/wet-3-1-culture-and-operation-of-a-caulinodans.pdf) |
| Knockout of the genome nifA gene in *A. caulinodans* | [Wet.3.2 Knockout of the genome nifA gene in *A. caulinodans*.pdf](https://static.igem.wiki/teams/4115/wiki/wet-3-2-knockout-of-the-genome-nifa-gene-in-a-caulinodans.pdf) |
| Validation of controlled expression of nitrogenase in *A. caulinodans* | [Wet.3.3 Validation of controlled expression of nitrogenase in *A. caulinodans*.pdf](https://static.igem.wiki/teams/4115/wiki/wet-3-3-validation-of-controlled-expression-of-nitrogenase-in-a-caulinodans.pdf) |
| Validation of intercellular signal response in *A. caulinodans* | [Wet.3.4 Validation of intercellular signal response in *A. caulinodans*.pdf](https://static.igem.wiki/teams/4115/wiki/wet-3-4-validation-of-intercellular-signal-response-in-a-caulinodans.pdf) |
| Preliminary Validation of Co-culture\_ Growth Curves | [Wet.4.1 Preliminary Validation of Co-culture\_ Growth Curves.pdf](https://static.igem.wiki/teams/4115/wiki/wet-4-1-preliminary-validation-of-co-culture-growth-curves.pdf) |
| Immobilization of microorganisms | [Wet.4.2 Immobilization of microorganisms.pdf](https://static.igem.wiki/teams/4115/wiki/wet-4-2-immobilization-of-microorganisms.pdf) |
| Co-culture of immobilized microorganisms | [Wet.4.3 Co-culture of immobilized microorganisms.pdf](https://static.igem.wiki/teams/4115/wiki/wet-4-3-co-culture-of-immobilized-microorganisms.pdf) |
| Intercellular communication in immobilized microorganisms | [Wet.4.4 Intercellular communication in immobilized microorganisms.pdf](https://static.igem.wiki/teams/4115/wiki/wet-4-4-intercellular-communication-in-immobilized-microorganisms.pdf) |

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the
engineering design cycle.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/4115/wiki/engineering-main2.jpeg)

![](https://static.igem.wiki/teams/4115/wiki/logowhite.svg)
![](https://static.igem.wiki/teams/4115/wiki/logowhite.svg)

Engineering

* [Overview](#1)
* [S. elongatus strains](#2)
* [Sucrose Production System](#3)
* [Nutrient Response](#4)
* [Nitrogen Fixation](#5)
* [Co-Culture in CoBG11](#6)
* [References](#7)

Overview

Our ternary microbial symbiosis system is consist of three chassis: *Escherichia coli*, *Synechococcus
elongatus* , and *Azotobacter caulinodans*. *S. elongatus* fix CO2 through
photosynthesis and secretes the product, sucrose as the carbon source for heterotrophic chassis. *A.
caulinodans* convert the N2 in Mars atmosphere into the universal nitrogen source,
NH4+. *E. coli* plays the role of production chassis. We performed a series of
engineering iterations to run sustainably on Mars. First, we have carefully considered engineering on *S.
elongatus* to maximize carbon source input. Second, we optimized nutrient response and nitrogen fixation
regulation part to allow our intercellular gene circuits to more efficiently support the ternary symbiotic
system. Finally, we present our optimization process on the medium composition and verify that all three strains
can grow in CoBG11.

Selection of *S. elongatus* strains

As the only autotrophic organism in our ternary microbial symbiosis system, *S. elongatus* is expected to
act as the direct carbon source supplier of *E. coli* and the indirect supplier of *A. caulinodans* in
our design. Based on the following two aspects, wild-type *S. elongatus* cannot meet our requirements. On
the one side, wild-type *S. elongatus* cannot secrete sucrose as a carbon source under normal conditions.
On the other side, the response pathway to signal molecules that are produced by *E. coli* when it is
hungry is deficient in wild-type *S. elongatus*. To overcome the two defects above, we need to perform
genetic manipulation on *S. elongatus*.

In order to find a suitable strain of *S. elongatus*, we first learned that *S. elongatus* PCC7942 and
UTEX2973 are constantly used for genetic transformation to study sucrose production and secretion based on
previous papers.[1, 2] PCC7942 is easy for gene manipulation but has a relatively slow growth rate.
By contraries, UTEX2973 has a relatively high growth rate but is difficult for gene manipulation. We then
contacted researcher Xuefeng Lv from Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy
of Sciences. We understood and obtained one strain called *S. elongatus* HL7942, an *S. elongatus*
strain modified from *S. elongatus* PCC7942. It retains the characteristics of easy gene manipulation and
has a higher growth rate than PCC7942 as well. Moreover, it has good genetic stability.

The gene manipulation of *S. elongatus* HL7942 can follow the natural transformation method. In other
words, plasmids can enter *S. elongatus* cells when we mix bacterial fluid and plasmid solution together.
In *S. elongatus* cells, plasmids cannot be copied. However, those who carry homologous arms of *S.
elongatus* genome can undergo homologous recombination with *S. elongatus* genome under natural
conditions. (Figure 1)

![](https://static.igem.wiki/teams/4115/wiki/engineering-recomb.png)

**Figure 1.** Gene recombination of *S. elongatus*

Based on the consideration above, we finally chose *S. elongatus* HL7942 as the strain for design and
experiment.

Sucrose Production System

Design

Sucrose permease (CscB) is a sucrose/proton symporter that can utilize proton gradients established across the
cell membrane to confer sucrose uptake activity specifically. Considering that most of the heterotrophs tend to
acidify the medium, which means *S. elongatus* is able to secrete sucrose through CscB, especially under
hypertonic conditions.[1] (Figure 2)

![](https://static.igem.wiki/teams/4115/wiki/engineering-mechcscb.png)

**Figure 2.** Schematic of sucrose permease(CscB) of *S. elongatus*

In order to simulate the regulatory effect of signal molecules on the expression of CscB, we set the promoter of
Cscb gene to lac promoter. In this way, IPTG can qualitatively simulate signal molecules, AHL, to raise the
expression of CscB. This will cause the concentration of sucrose in the culture medium to increase. In addition,
to select the bacteria that successfully integrate the target fragment, we also designed kanamycin resistance
(KanR) gene on the integrated fragment ([BBa\_K4115045](http://parts.igem.org/Part%3ABBa_K4115045)).

![](https://static.igem.wiki/teams/4115/wiki/engineering-plasmidcscb.png)

**Figure 3.** Construction of pUC57-NS3-2-Plac\_UV5-CscB-lacI-KanR-NS3-2

Build

After the transformation and successful selection of recombinant *S. elongatus*, we did bacterial colony
PCR to prove that the target fragments, especially CscB gene and LacI gene, were integrated into the *S.
elongatus* genome. (Figure 4) Then the target bands were recovered for sequencing. (Figure 5)

![](https://static.igem.wiki/teams/4115/wiki/engineering-pcrcscb.png)

**Figure 4.** Colony PCR of recombinant *S. elongatus* at different annealing temperature. The area
shown in the red box is the target band area. As the annealing temperature rises, non-specific binding
reduces. The upper and lower figures are cut from the same gel map for easy annotation and observation.

![](https://static.igem.wiki/teams/4115/wiki/engineering-checkcscb.png)

**Figure 5.** Sequencing results showed that there was no mutation in CscB gene and LacI gene.

Test

After expanding the culture of recombinant *S. elongatus*, we set four different cultivation conditions to
culture *S. elongatus* and recombinant *S. elongatus*. (Table 1) We compared the accumulation of
sucrose secreted by a unit number of *S. elongatus* in a certain period of time by comparing the ratio of
sucrose concentration in the supernatant to OD685 value of *S. elongatus*. (Figure 6)

|  | **Group C-1** | **Group C-2** | **Group C-3** | **Group C-4** | **Group E-1** | **Group E-2** | **Group E-3** | **Group E-4** |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| **Wild-type *S. elongatus* fluid (μl)** | 5000 | 4995 | 4500 | 4495 | 0 | 0 | 0 | 0 |
| ***S. elongatus* transformed with cscB gene fluid (μl)** | 0 | 0 | 0 | 0 | 5000 | 4995 | 4500 | 4495 |
| **1M IPTG solution (μl)** | 0 | 5 | 0 | 5 | 0 | 5 | 0 | 5 |
| **1M NaCl solution (μl)** | 0 | 0 | 500 | 500 | 0 | 0 | 500 | 500 |
| **Final volume of fluid (ml)** | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
| **Number of parallel groups** | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 |

**Table 1.** Composition of control groups and experimental groups in sucrose production detection
experiment.

![](https://static.igem.wiki/teams/4115/wiki/engineering-induced-24h.png)

**Figure 6.** The value of g/(L·OD685) of different groups after inducing 24 hours.

Learn

We found that under the condition that CscB and NaCl exist together, the effect of sucrose production by *S. elongatus* is the best. This fact indicates that adding a certain concentration of NaCl to the co-culture medium has a positive effect on our ternary microbial symbiosis system.

Optimization of Nutrient Response

We construct intercellular genetic circuit feedback to regulate the nutrient flux in our ternary microbial symbiosis system. In the genetic circuit, starvation promoters in *E. coli* are sensors for nutrient signals. To ensure a good signal-to-noise ratio for signal input, we made efforts on improving the properties of the starvation promoters.

We evaluate the quality of promoters from **two aspects**. First, **promoter activity** is an important evaluation index. Empirically, we believe that promoter activity not lower than the J23101 (a constitutive promoter with moderately strong activity) is necessary for production and genetic circuits. Second, **fold-change** is another important perspective for promoters, especially for those being used in complicated genetic circuits. Fold-change can be defined as the ratio of the activity in the activated state to the non-activated state. For our starvation promoters, the practical definition is the ratio of promoter activity under low glucose concentration to that under high glucose concentration.

We first selected three starvation promoters and test their response to low glucose concentrations. We construct reporter genes as the construct in Figure 7, and quantified the promoter activity with fluorescence intensity. Among the three promoters, PcstA had the largest fold-change and was comparable to the activity of J23101. However, compared with the widely used inducible promoters like pLac or pBAD, its activity and fold-change are not good enough. So we did several improvement cycles based on the 'Design-Build-Test-Learn' cycle.

![](https://static.igem.wiki/teams/4115/wiki/engineering-reporter.png)

**Figure 7.** sfGFP with LVA is used to indicate the activities of PcstA variants.

First cycle

PcstA ([BBa\_K118011](http://parts.igem.org/Part%3ABBa_K118011)) is a well-characterized starvation promoter in iGEM Registry. PcstA uses RpoD as its sigma factor. cAMP receptor protein (CRP) is an activator of PcstA. When undergoes carbon source starvation conditions, the intracellular cAMP concentration will increase and further activate CRP. Then CRP binds to a CRP-binding sequence located upstream of the transcription start site and activates of transcription initiation of PcstA (Figure 8).

![](https://static.igem.wiki/teams/4115/wiki/engineering-mechanism.png)

**Figure 8.** CRP-dependent activation of PcstA under starvation conditions.

Furthermore, PcstA is down-regulated by the factor for inversion stimulation (Fis) under nutrient-rich conditions (Figure 9). Under starvation conditions, the Fis abundance in cells will decrease, and the inhibition will be removed.

![](https://static.igem.wiki/teams/4115/wiki/engineering-fis.png)

**Figure 9.** Fis-dependent inhibition of PcstA under nutrient-rich conditions.

To further optimize the activity and fold-change of PcstA, we made several mutations in its regulation-related sequence. We first mutate the CRP-binding sequence to improve the affinity of the promoter for CRP, so that CRP can activate the promoter more efficiently. The mutants constructed in the first cycle are shown in Figure 4. In principle, if the CRP-binding sequence of PcstA has higher similarity to the consensus sequence, PcstA will have a higher affinity to CRP. The consensus sequence of the CRP-binding site is AAATGTGA-N6-TCTCATTT. The nucleotides with underline are the core of the CRP-binding sequence. G and C with underlines provide most of the binding energy[4]. Based on these rules, we constructed PcstA-Con and PcstA-TCACA to test what will happen if PcstA has a higher affinity to CRP.

![](https://static.igem.wiki/teams/4115/wiki/engineering-first-cycle.png)

**Figure 10.** The CRP-binding sequences of PcstA variants in the first cycle.

The reporter gene in Figure 1 is used to test the properties of these PcstA variants. Data is summarized in Figure 11. The promoter activity was significantly improved by the mutations. PcstA-Con has a 15 times higher activity compared with the original PcstA. So it's hard to indicate the data in a column figure. However, it seems that their affinities to CRP are too high. Even under a high glucose concentration of 4 g/L, they are still activated and show no response to starvation. In this cycle, we learnt that high similarity with the consensus sequence is not always a good thing. There might be a suitable range for the affinities of regulators.

![](https://static.igem.wiki/teams/4115/wiki/engineering-first-cycle-data.png)

**Figure 11.** Summarized data of the relative activities and fold-changes of PcstA variants. The relative activities are normalized with FI/OD600 of J23101 at 4g/L glucose.

Second cycle

Then we decide to mutate another site that has relatively little effect on affinity.

![](https://static.igem.wiki/teams/4115/wiki/engineering-second-cycle.png)

**Figure 12.** The CRP-binding sequences of PcstA variants in the second cycle.

The same conditions and analysis method were used to test the property of PcstA\_Mutant1. The reporter gene constructs are cloned into pUC high-copy number backbone. A significant promoter activity increase was observed on PcstA\_Mutant1 and the starvation response function remained (Figure 13A). However, some unwanted phenomena also appeared. First, the leakage expression of PcstA\_Mutant1 under high glucose concentration increases, which results in the decrease of fold-change. Second, the high copy of PcstA\_Mutant1 in cells has some side effects on growth (Figure 13B). Actually, the growth rate decrease was more obvious in PcstA-Con and PcstA-TCACA transformed strains. So we hypothesize that the high copy of PcstA\_Mutant1 causes these side effects. If PcstA\_Mutant1 keeps a high copy in cells, it may compete the CRP with the endogenous CRP-dependent promoter and influence the global metabolism.

![](https://static.igem.wiki/teams/4115/wiki/engineering-mutant1.png)
![](https://static.igem.wiki/teams/4115/wiki/engineering-growth-decrease.png)

**Figure 13.** (A) FI/OD600 of PcstA and PcstA\_Mutant1 (B) Growth curve of Empty and PcstA\_Mutant1 strain in M9 minimal medium containing 4.0 g/L glucose as the sole carbon source.

Third cycle

At the end of the last cycle, we got a function variant, which has higher activity and remains the starvation response. To eliminate the undesirable properties of PcstA\_Mutant1, we shifted all constructions to the low-copy number pET backbone and repeat the experiment (Figure 14). The bacteria growth is back to normal in the PcstA\_Mutant1 group, which may partially support our hypothesis on the growth. Mutants of PcstA show significantly higher activities as well as larger fold-changes with low-copy number backbone. The data of relative promoter activities and fold changes of PcstA variants in this cycle are summarized in Figure 15 .

![](https://static.igem.wiki/teams/4115/wiki/engineering-low-copy.png)

**Figure 14.** FI/OD (600) of PcstA and PcstA\_Mutant1. All the constructs are cloned into pET low-copy number backbone.

In conclusion, by mutating the regulation-related sequence and replacing the plasmid backbone, we finally get Pcst\_Mutant1 (on pET backbone) as the best construct for the nutrient response.

![](https://static.igem.wiki/teams/4115/wiki/engineering-conclusion.png)

**Figure 15.** Summarized data of PcstA variants. High [Glucose]=2 g/L, Low [Glucose]=0.25 g/L. Relative promoter activities are normalized with FI/OD (600) of J23101 at 2 g/L glucose

Controllable regulation of nitrogen fixation rate

In *A. caulinodans*, the nitrogen fixation is tightly controlled by the concentration of intracellular ammonium and this counts for the nitrogenase master regulator *nifA*[7]. To achieve controllable regulation of nitrogen fixation rate, endogenous regulators must be removed. In previous research, knocking out of genomic *nifA* gene and then complementing by *nifA* controlled by inducible promoters was proved to be a viable strategy[5, 6]. So we first transformed the suicide plasmid into *A. caulinodans* to construct the *ΔnifA* strain, and then we transformed the shuttle plasmid into the *ΔnifA* strain to complement the inducible *nifA* gene. In the experimental process, we successively carried out two rounds of engineering optimization, finally designed a successful strategy, and completed the verification experiment of the *nifA* knockout. We learned from research that the knockout of a target gene can be achieved by inserting a DNA sequence into a specific site on the genome using homologous recombination. So we synthesized the 900 bp sequence upstream and downstream of *nifA* on the genome as the homologous arm (upsteam-homologous-arm, UHA & downsteam-homologous-arm, DHA).

In **strategy-one**, the inducible *nifA* fragment (*Plac-nifA-ChlR-LacI*) containing the *ChlR* gene, a chloramphenicol resistance marker, was designed to be reversely integrated into the genome (the reintegrated *nifA* is in the opposite direction to the genomic *nifA*). In this way, the knockout and inducible complement of *nifA* could be realized in on step. However, this strategy turned out to failed in PCR validation. Failure to integrate fragments into the genome may be due to excessive length or interference by homologous recombination (*nifA* itself can also act as a homologous arm). Later we found that it was more likely that the resistance tag failed (seen in **strategy-two** ).

In **strategy-two**, only the *ChlR* gene was designed to be integrated into the genome to knock out the *nifA*. However, this strategy turned out to failed in PCR validation as well. In subsequent PCR and sequencing analysis, we found that our *A. caulinodans* ORS571 strain contained ChlR gene on its genome, which which is the information we didn't get through research and inquiry before. Therefore, we concluded that the former two strategies failed due to the incorrect resistance marker.

In **strategy-three**, the *ChlR* gene was replaced by bleomycin resistance gene *BleoR*. And the the inducible *nifA* fragment was constructed in pBBR1 plasmid with the report fragment (*PnifH-sfgfp*). Finally, the *ΔnifA* strain was constructed and validated (seen in [Result](https://2022.igem.wiki/shanghaitech-china/results)). Therefore, we designed the suicide plasmid ([BBa\_K4115041](http://parts.igem.org/Part%3ABBa_K4115041)), report shuttle plasmid ([BBa\_K4115042](http://parts.igem.org/Part%3ABBa_K4115042)) and inducible and report plasimid ([BBa\_K4115043](http://parts.igem.org/Part%3ABBa_K4115043)).

![](https://static.igem.wiki/teams/4115/wiki/engineering-strategys.png)

**Figure 16.** Three strategies in controllable regulation of nitrogen fixation rate. (a) Strategy-one; (b) Strategy-two; (c) Strategy-three.

The gel result of the PCR validation preliminarily verified the success of knockout. Further sequencing validation has proved that the construction of *ΔnifA* strain was completed. More detials and analysis is on [Results](https://2022.igem.wiki/shanghaitech-china/results) page.

![](https://static.igem.wiki/teams/4115/wiki/results-gel-knockout.png)

**Figure 17.** Colony PCR of *ΔnifA* colony.

Co-Culture in the CoBG11 Medium

The separate-immobilized fermentation pattern we proposed should be how the three microbial symbiotic system is implemented in practice. To prove this concept, the three microorganisms should survive under some of the same medium condition. We first set up a medium condition in which three microorganisms could separately live.

We had known from previous research about the CoBG11 medium protocols for the coculture of Synechococcus and other heterotrophic microorganisms. Based on this, we proposed our CoBG11-Sucrose medium protocols by adding sucrose as the sole carbon source to simulate the sucrose secretion of *S. elongatus*. Moreover, ammonium chloride in the medium is used as a simulation of ammonium production by *A. caulinodans*.

![](https://static.igem.wiki/teams/4115/wiki/results-separate1.png)

**Figure 18.** The growth curve of three microorganisms in CoBG11-Sucrose medium.

Through the resulting growth curve, we found that both *E. coli* and *S. elongatus* can live and grow in CoBG11-Sucrose medium (Figure 18a&b). However, *A. caulinodans* quickly reached a stationary phase and remained at a low optical density, which shows that it cannot grow in CoBG11-Sucrose medium (Figure 18.c). After research review, our hypothesis was confirmed. *A. caulinodans* prefers to use organic acid as its carbon source rather than sugar[8]. Therefore, when the medium contains only sugar as solo carbon source, *A. caulinodans* cannot live and grow.

We found that *E. coli* would excrete significant amounts of acetate when growing aerobically on glucose as the sole carbon source, which SacC-positive *E. coli* growing on sucrose will be in a similar situation[9]. In this case, *A. caulinodans* can take acetate as its solo carbon source. Therefore, we proposed a new CoBG11-Acetate protocol by adding acetate as solo carbon source to simulate the acetate secretion of *E. coli*. In this experiment, we culture the *A. caulinodans* both in CoBG11-Sucrose and CoBG11-Acetate to prove that the change of carbon sources can make *A. caulinodans* successfully live in CoBG11 medium.

![](https://static.igem.wiki/teams/4115/wiki/results-carbon.png)

**Figure 19.** The growth curve of *A. caulinodans* in CoBG11-Sucrose/Acetate medium.

After replacing the carbon source, the growth of *A. caulinodans* was significantly improved. The result confirms that *A. caulinodans* can live and grow in CoBG11 medium when acetate is the solo carbon source.

Reference

[1] Ducat, D.C., Avelar-Rivas, J.A., Way, J.C., and Silver, P.A. (2012). Rerouting carbon flux to enhance photosynthetic productivity. Appl Environ Microbiol *78*, 2660-2668.

[2] Song, K., Tan, X., Liang, Y., and Lu, X. (2016). The potential of Synechococcus elongatus UTEX 2973 for sugar feedstock production. Appl Microbiol Biotechnol *100*, 7865-7875.

[3]Lin, P.C., Zhang, F., and Pakrasi, H.B. (2020). Enhanced production of sucrose in the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973. Sci Rep *10*, 390.

[4] Soberon-Chavez, G., Alcaraz, L.D., Morales, E., Ponce-Soto, G.Y., and Servin-Gonzalez, L. (2017). The Transcriptional Regulators of the CRP Family Regulate Different Essential Bacterial Functions and Can Be Inherited Vertically and Horizontally. Front Microbiol 8. ARTN 959
10.3389/fmicb.2017.00959.

[5] Ryu, M.H., Zhang, J., Toth, T., Khokhani, D., Geddes, B.A., Mus, F., Garcia-Costas, A., Peters, J.W., Poole, P.S., Ané, J.M., et al. (2020). Control of nitrogen fixation in bacteria that associate with cereals. Nat Microbiol 5, 314-330.

[6] Haskett, T.L., Paramasivan, P., Mendes, M.D., Green, P., Geddes, B.A., Knights, H.E., Jorrin, B., Ryu, M.-H., Brett, P., Voigt, C.A., et al. (2022). Engineered plant control of associative nitrogen fixation. Proceedings of the National Academy of Sciences 119, e2117465119.

[7] Demtröder, L., Pfänder, Y., Schäkermann, S., Bandow, J.E., and Masepohl, B. (2019). *nifA* is the master regulator of both nitrogenase systems in Rhodobacter capsulatus. Microbiologyopen 8, e921.

[8] 郝保海 (2012). 根瘤菌比较基因组学研究及蛋白质互作网络预测 (华中农业大学).

[9] Bernal, V., Castaño-Cerezo, S., and Cánovas, M. (2016). Acetate metabolism regulation in Escherichia coli: carbon overflow, pathogenicity, and beyond. Applied Microbiology and Biotechnology 100, 8985-9001.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/4115/wiki/experiment-main.jpg)

![](https://static.igem.wiki/teams/4115/wiki/logowhite.svg)
![](https://static.igem.wiki/teams/4115/wiki/logowhite.svg)

Experiments

* [Culture Medium
  Preparation](#1)
* [Bacterial Strain
  Recovery](#2)
* [Bacterial Growth
  Detection](#3)
* [Bacterial Culture](#4)
* [Bacterial
  Cryopreservation](#5)
* [Plasmid construction](#6)
* [Experiments on S.
  elongatus](#7)
* [Experiments on E.
  coli](#8)
* [Experiments on A.
  caulinodans](#9)
* [Co-Culture](#10)

| **List of abbreviation** | |
| --- | --- |
| **Abbreviation** | **Full Name** |
| IPTG | Isoprophyl-β-D-thiogalactoside |
| HEPES | 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid |
| HEPPS | 4-Hydroxyerhylpiperazine-1-propanesulfonic acid |
| EDTA | Ethylenediaminetetraacetic acid |
| kb | Kilo base pairs |
| DNA | Deoxyribonucleic acid |
| OD | Optical density |
| PCR | Polymerase chain reaction |
| EP tube | Graduated tip bottom centrifuge tube |
| TY | Tryptone-Yeast extract |
| rpm | Revolutions per minute |
| *E. coli* | *Escherichia coli* |
| *S. elongatus* | *Synechococcus elongatus HL7942* |
| *A. caulinodans* | *Azorhizobium caulinodans ORS571* |
| TAE buffer | Buffer solution containing trihydroxymethyl aminomethane, acetic acid, and ethylenediamine tetraacetic acid of certain concentration |
| CscB | Sucrose permease |
| Kan | Kanamycin |
| Cm | Chloramphenicol |
| Amp | Ampicillin |
| Ble | Bleomycin |

Culture Medium Preparation

BG11

BG11 liquid medium

**Reagent**: BG11 medium powder(hopebio, product number: HB8793), Milli-Q water

**Device**: Measuring cylinder, Electronic balance, Medicine spoon

Procedure(for a 1L mixture):

1. Accurately weigh 1.7 g of BG11 medium powder and then add 1 L of Milli-Q water.
2. High-pressure steam sterilization at 121℃ for 15 min.
3. If the non-resistant medium is required, use it after cooling. If the resistant medium is required, add
   antibiotics after cooling.
   Make sure to use it after mixing completely.

**P.S.** Antibiotic concentration of *S. elongatus* selection is as follows: Kan 20 ng/μl, Cm 10 ng/μl.
Notice that *Synechococcus elongatus* HL7942 has basic resistance to ampicillin.

BG11 plate

**Reagent**: BG11 medium powder(hopebio, product number: HB8793), Milli-Q water, Agar powder,
NaS2O3·5H2O

**Device**: Measuring cylinder, Electronic balance, Medicine spoon

Procedure(for a 1 L mixture with 2% agar):

1. Accurately weigh 1.7 g of BG11 medium powder, 4.8 g NaS2O3·5H2O, and 20.0 g
   agar powder. Mix them and then add 1 L of Milli-Q water.
2. High-pressure steam sterilization at 121℃ for 15 min.
3. If the non-resistant medium is required, pour the plate before solidification. If the resistant medium is
   required, add antibiotics after cooling but before solidification.
4. Make sure to mix well before pouring. Pour the plate until the bottom of the petri dish is completely
   covered and the thickness reaches 2-3 mm.

**P.S.** Lack of NaS2O3·5H2O will reduce the hardness of the culture medium.

|  |  |
| --- | --- |
| **List of ingredients for BG11 medium powder(hopebio, product number: HB8793)** | |
| **Component** | **Content (g/L)** |
| NaNO3 | 1.5 |
| K2HPO4·3H20 | 0.04 |
| MgSO4·7H2O | 0.075 |
| CaCl2·2H2O | 0.036 |
| Citric acid | 0.006 |
| Ferric ammonium citrate | 0.006 |
| EDTA | 0.001 |
| NaCO3 | 0.02 |
| H3BO3 | 0.00286 |
| MnCl2·H20 | 0.00181 |
| ZnSO4·7H2O | 0.000222 |
| CuSO4·5H2O | 0.000079 |
| Na2MoO4·2H2O | 0.00039 |
| Co(NO3)2·6H2O | 0.000049 |

LB

LB liquid medium

**Reagent**: LB broth powder(Sangon Biotech, product number: A507002), Milli-Q water

**Device**: Measuring cylinder, Electronic balance, Medicine spoon

Procedure(for a 1 L mixture):

1. Accurately weigh 25 g of LB broth powder and then add 1 L of Milli-Q water.
2. High-pressure steam sterilization at 121℃ for 15 min.
3. If the non-resistant medium is required, use it after cooling. If the resistant medium is required, add
   antibiotics after cooling. Make sure to use it after mixing completely.

**P.S.** Antibiotic concentration of *E. coli* selection is as follows: Kan 30 ng/μl, Cm 34 ng/μl, and
Amp 100 ng/μl.

|  |  |
| --- | --- |
| **List of ingredients for LB broth powder(Sangon Biotech, product number: A507002)** | |
| **Component** | **Content (g/L)** |
| Tryptone | 10 |
| Yeast extract | 5 |
| NaCl | 10 |

LB plate

**Reagent**: LB broth Agar powder(Sangon Biotech, product number: A507003), Milli-Q water

**Device**: Measuring cylinder, Electronic balance, Medicine spoon

Procedure(for a 1 L mixture with 1.5% agar):

1. Accurately weigh 40 g of LB broth agar powder and then add 1 L of Milli-Q water.
2. High-pressure steam sterilization at 121℃ for 15 min.
3. If the non-resistant medium is required, pour the plate before solidification. If the resistant medium is
   required, add antibiotics after cooling but before solidification.
4. Make sure to mix well before pouring. Pour the plate until the bottom of the petri dish is completely
   covered and the thickness reaches 2-3 mm.

|  |  |
| --- | --- |
| **List of ingredients for M9 broth powder(Sangon Biotech, product number: A507024)** | |
| **Component** | **Content (g/L)** |
| Na2HPO4 | 6.8 |
| KH2PO4 | 3 |
| NaCl | 0.5 |
| NH4Cl | 1 |

M9

M9 minimal liquid medium with sucrose

**Reagent**: M9 broth powder(Sangon Biotech, product number: A507024), 1 M MgSO4 solution, 1 M
CaCl2 solution, Milli-Q water, 20% Sucrose solution

**Device**: Measuring cylinder, Electronic balance, Medicine spoon, BeyoGold Syringe Filter (0.22μm/3mm, PES,
Sterile)

Procedure(for a 100 ml mixture):

1. Accurately weigh 1.13 g of M9 broth powder and then add 95 ml of Milli-Q water.
2. Sterilize M9 salts solution, MgSO4 solution, and CaCl2 by high-pressure steam
   sterilization at 121℃ for 15 min respectively. Sterilize 20% sucrose solution by a filter.
3. After cooling, mix 95 ml of M9 salts solution, 200 μl of 1 M MgSO4 solution, 10 μl of 1 M
   CaCl2 solution, and 5 ml of 20% sucrose solution.
4. If the resistant medium is required, add antibiotics after cooling. Make sure to use it after mixing
   completely.

|  |  |
| --- | --- |
| **List of ingredients for LB broth agar powder(Sangon Biotech, product number: A507003)** | |
| **Component** | **Content (g/L)** |
| Tryptone | 10 |
| Yeast extract | 5 |
| NaCl | 10 |
| Agar | 15 |

M9 minimal liquid medium with glucose

**Reagent**: M9 broth powder(Sangon Biotech, product number: A507024), 1 M MgSO4, 1 M
CaCl2, Milli-Q water, glucose

**Device**: Measuring cylinder, Electronic balance, Medicine spoon
Procedure(for a 1 L mixture):

1. Accurately weigh 11.30 g of M9 broth powder and then add 978 ml of Milli-Q water.
2. Sterilize M9 salts solution, MgSO4 solution, and CaCl2 by high-pressure steam sterilization at 121℃ for 15 min respectively.
3. Prepare glucose solutions of different concentrations and sterilize them by filtration.
4. After cooling, mix 978 ml of M9 salts solution, 20 ml of glucose solution, 2 ml of 1 M MgSO4 solution, 100 μl of 1 M CaCl2 solution.
5. If the resistant medium is required, add antibiotics after cooling. Make sure to use it after mixing completely.

TY

TY liquid medium

**Reagent**: Tryptone, Yeast extract, CaCl2, Milli-Q water

**Device**: Measuring cylinder, Electronic balance, Medicine spoon

Procedure(for a 1 L mixture):

1. Accurately weigh 5.0 g tryptone, 3.0 g yeast extract, and 0.6 g CaCl2. Then add 1 L of Milli-Q
   water.
2. High-pressure steam sterilization at 121℃ for 20 min.
3. If the non-resistant medium is required, use it after cooling. If the resistant medium is required, add
   antibiotics after cooling. Make sure to use it after mixing completely.

**P.S.** Antibiotic concentration of *A. caulindans* selection is as follows: Ble 100 ng/μl, and Amp 100
ng/μl.

TY plate

**Reagent**: Tryptone, Yeast extract, CaCl2, Milli-Q water, Agar powder

**Device**: Measuring cylinder, Electronic balance, Medicine spoon

Procedure(for a 1 L mixture with 2% agar):

1. Accurately weigh 5.0 g tryptone, 3.0 g yeast extract, 20.0 g agar powder, and 0.6 g CaCl2. Then
   add 1L of Milli-Q water.
2. High-pressure steam sterilization at 121℃ for 20 min.
3. If the non-resistant medium is required, pour the plate before solidification. If the resistant medium is
   required, add antibiotics after cooling but before solidification.
4. Make sure to mix well before pouring. Pour the plate until the bottom of the petri dish is completely
   covered and the thickness reaches 2-3 mm.

CoBG11

CoBG11 liquid medium

**Reagent**: BG11 medium powder(hopebio, product number: HB8793), NH4Cl, NaCl, HEPES, Milli-Q
water

**Device**: Measuring cylinder, Electronic balance, Medicine spoon, BeyoGold Syringe Filter (0.22μm/3mm,
PES, Sterile)

Procedure(for a 1 L mixture):

1. Accurately weigh 1.700 g of BG11 medium powder, 6.201 g NaCl, 0.214 g NH4Cl, and 5.958 g HEPES.
   Then add 1 L of Milli-Q water. Make the final concentration of NaCl reach 106mM, NH4Cl reach 4mM,
   HEPES reach 25mM. Adjust the pH to 7.5.
2. Use the filter to filter the solution or sterilize by high-pressure steam sterilization at 121℃ for 15 min.
3. If the non-resistant medium is required, use it after cooling. If the resistant medium is required, add
   antibiotics after cooling. Make sure to use it after mixing completely.

Bacterial Strain Recovery

*E. coli* and *S. elongatus*

**Reagent**: Glycerol bacteria preserved at -40℃ or -80℃, Ice, Non-resistant liquid medium corresponding to
the bacteria strain

**Device**: Icebox, Ultra clean bench, Culture tubes, Pipette gun, Sterile pipette tips, Refrigerator

Procedure:

1. Take 3-5ml non-resistant liquid medium on the ultra clean bench and put it into the culture tube.
2. Remove the glycerol bacteria from the -40℃ or -80℃ refrigerator and immediately put it into the icebox
   containing ice.
3. Quickly remove the ice residue of glycerol bacteria on the ultra clean bench by the pipette tip and put it
   into the culture medium.
4. Quickly put the glycerol bacteria back into the refrigerator before melting.
5. Label the culture tube and put it into the shaker. Culture until the solution turns turbid.

**P.S.** Avoid repeated freezing and thawing.

*A. caulinodans*

**Reagent**: Glycerol bacteria preserved at -40℃ or -80℃, Ice, Non-resistant TY liquid medium, Resistant TY
plate.

**Device**: Icebox, Ultra clean bench, Culture tubes, Pipette gun, Sterile pipette tips, Refrigerator,
NanoDrop One

Procedure:

1. Inoculate 50 μl glycerol bacteria into 5 ml TY liquid medium and culture for 2 ~ 3 days.
2. Perform streak plate method on TY solid medium containing 100 μg/ml ampicillin and culture at 37℃ for 2
   days.
3. Inoculate single colonies into 4 ml TY liquid medium containing 100 μg/ml ampicillin and culture until
   OD600 = 0.5 ~ 0.7.

Bacterial Growth Detection

**Reagent**: Bacterial fluid to be tested, Liquid medium corresponding to the sample, Double distilled water

**Device**: Ultra clean bench, Pipette gun, Pipette tips, Sterile pipette tips, NanoDrop one ultramicro
ultraviolet spectrophotometer(Thermo SCIENTIFIC), Lens papers

Procedure:

1. Open NanoDrop one in advance.
2. Take out the sample of bacterial fluid to be tested on the ultra clean bench.
3. Select the "OD600 " tab and click the "OD600" option on the home page of NanoDrop one.
4. Fill in "other detection wavelengths" according to the type of bacteria to be tested. For *E. coli* or
   \*A. caulinodans\*, fill in 600 or not. For *S. elongatus*, fill in 685.
5. Clean the instrument base with double distilled water. Wipe clean with lens papers.
6. Use 1 μl of liquid medium corresponding to the sample as a blank sample. After detecting, clean the
   instrument base with double distilled water. Wipe clean with lens papers.
7. Add 1 μl bacterial fluid to the base for detection. After detecting, record data and clean the instrument
   base with double distilled water. Wipe clean with lens papers.
8. Shut down the instrument after use.

**P.S.** Complete mixing is required before sampling and loading of *S. elongatus* bacterial fluid.

Microplate Reader

**Reagent**: Bacterial fluid to be tested, Liquid medium corresponding to the sample

**Device**: Ultra clean bench, Pipette gun, Sterile pipette tips, Microplate Reader(Thermo SCIENTIFIC),
96-well plate(or disposable enzyme label strips and enzyme label plate)

Procedure:

1. Open the microplate reader in advance.
2. Add 200μl of bacterial fluid to be tested into 96-well plate(or disposable enzyme label strips and enzyme
   label plate) on the ultra clean bench. Also add 200 μl of liquid medium corresponding to the sample as a blank
   sample.
3. Put the 96-well plate(or enzyme label plate) into the microplate reader. Select detection area and
   wavelength. Then click "start".
4. Record the data after detecting. The OD value of the sample is the sample detection value minus the blank
   sample detection value.
5. Shut down the microplate reader after use.

Bacterial Culture and Inoculation

*Escherichia coli*

Liquid

**Culture**

Conditions: 37℃, 220 rpm, no special requirements for lighting.

**Inoculation**

**Reagent**: LB liquid medium, Bacterial fluid

**Device**: Ultra clean bench, Pipette gun, Sterile pipette tips, Culture tubes, Thermostatic shaker

Procedure:

1. Take bacterial fluid in the logarithmic growth period(OD600=0.5-0.6).
2. Inoculate the bacterial fluid to the fresh medium at a ratio of 1:100 in the culture tube.
3. Label and put the tube in the thermostatic shaker.

**P.S.** Steps 1-2 operate on the ultra clean bench

Plate

**Culture**

Conditions: 37℃, no special requirements for lighting.

**Inoculation**

**Reagent**: LB plate, Bacterial fluid

**Device**: Ultra clean bench, Pipette gun, Sterile pipette tips, Thermostatic incubator, Disposable
spreaders, Disposable inoculation loops, Parafilm

Procedure:

**Spread Plate Method**

1. Remove 100 μl bacterial fluid to the LB plate.
2. Use a disposable spreader to evenly coat the bacterial fluid on the LB plate. Keep coating until no flowing
   liquid on the surface of the plate.
3. Seal the plate with parafilm. Label and put the plate in the thermostatic incubator.

**P.S.** Steps 1-2 operate on the ultra clean bench

**Streak Plate Method**

1. Dip the bacterial fluid by a disposable inoculation loop.
2. Streak the LB plate by the inoculation loop with bacterial fluid drop.
3. Seal the plate with parafilm. Label and put the plate in the thermostatic incubator.

**P.S.** Steps 1-2 operate on the ultra clean bench

*Synechococcus elongatus HL7942*

Liquid

**Culture**

Conditions: 30℃, 130 rpm, 10 W lamp tube.

**Inoculation**

**Reagent**: BG11 liquid medium, Bacterial fluid

**Device**: Ultra clean bench, Pipette gun, Sterile pipette tips, Culture tubes, Thermostatic shaker with
external lamp tube

Procedure:

1. Take bacterial fluid in the logarithmic growth period(OD685≈0.5).
2. Inoculate the bacterial fluid to the fresh medium at a ratio of 1:100 in the culture tube.
3. Label and put the tube in the Thermostatic shaker with an external lamp tube.

**P.S.** Steps 1-2 operate on the ultra clean bench

Plate

**Culture**

Conditions: 30℃, 8000 lux.

**Inoculation**

**Reagent**: BG11 plate, Bacterial fluid

**Device**: Ultra clean bench, Pipette gun, Sterile pipette tips, Thermostatic light incubator, Disposable
spreaders, Parafilm

Procedure:

1. Remove 100 μl bacterial fluid to the BG11 plate.
2. Use a disposable spreader to evenly coat the bacterial fluid on the BG11 plate. Keep coating until no
   flowing liquid on the surface of the plate.
3. Seal the plate with parafilm. Label and put the plate in the thermostatic light incubator.

**P.S.** Steps 1-2 operate on the ultra clean bench

*Azorhizobium caulinodans ORS571*

Liquid

**Culture**

Conditions: 37℃, 180 rpm, no special requirements for lighting.

**Inoculation**

**Reagent**: TY liquid medium, Bacterial fluid

**Device**: Ultra clean bench, Pipette gun, Sterile pipette tips, Culture tubes, Thermostatic shaker

Procedure:

1. Take bacterial fluid in the logarithmic growth period(OD600=0.5-0.6).
2. Inoculate the bacterial fluid to the fresh medium at a ratio of 1:100 in the culture tube.
3. Label and put the tube in the thermostatic shaker.

**P.S.** Steps 1-2 operate on the ultra clean bench

Plate

**Culture**

Conditions: 37℃, no special requirements for lighting.

**Inoculation**

**Reagent**: TY plate, Bacterial fluid

**Device**: Ultra clean bench, Pipette gun, Sterile pipette tips, Thermostatic incubator, Disposable
spreaders, Disposable inoculation loops, Parafilm

Procedure:

**Spread Plate Method**

1. Remove 100 μl bacterial fluid to the TY plate.
2. Use a disposable spreader to evenly coat the bacterial fluid on the TY plate. Keep coating until no flowing
   liquid on the surface of the plate.
3. Seal the plate with parafilm. Label and put the plate in the thermostatic incubator.

**P.S.** Steps 1-2 operate on the ultra clean bench

**Streak Plate Method**

1. Dip the bacterial fluid by a disposable inoculation loop.
2. Streak the TY plate by the inoculation loop with bacterial fluid drop.
3. Seal the plate with parafilm. Label and put the plate in the thermostatic incubator.

**P.S.** Steps 1-2 operate on the ultra clean bench

Bacterial Cryopreservation

*E. coli*

**Reagent**: bacterial fluid to be frozen, Sterile 50% glycerol, -80℃ Refrigerator

**Device**: Ultra clean bench, Pipette gun, Sterile pipette tips, 1.5 ml EP tube

Procedure(for 500 μl bacterial fluid):

1. Take 500 μl of bacterial fluid whose OD600 reaches 0.5.
2. Mix 500 μl bacterial fluid with 500 μl sterile 50% glycerol together in a 1.5 ml EP tube. Make the final
   concentration of glycerol reach 25%.
3. Label and put the EP tube into the -80℃ refrigerator.

**P.S.** Operate on the ultra clean bench

*S. elongatus*

**Reagent**: bacterial fluid to be frozen, Sterile 50% glycerol, Non-resistant BG11 liquid medium, -80℃
Refrigerator

**Device**: Ultra clean bench, Pipette gun, Sterile pipette tips, Centrifuge, Centrifuge tube, 1.5 ml EP
tube

Procedure(for 4 ml bacterial fluid):

1. Take 4 ml of bacterial fluid whose OD685 reaches 0.5 in the suitable centrifuge tube.
2. Centrifuge to collect bacteria at 6000 g at 4℃ for 10 min.
3. Discard the supernatant and use 2 ml of non-resistant BG11 liquid medium for resuspension. Centrifuge to
   collect bacteria at 6000 g at 4℃ for 10 min.
4. Repeat Step 3.
5. Discard the supernatant and use 800 μl of non-resistant BG11 liquid medium for resuspension.
6. Mix 680 μl concentrated liquid with 320 μl sterile 50% glycerol together in a 1.5 ml EP tube. Make the final
   concentration of glycerol reach 16%.
7. Label and put the EP tube into the -80℃ refrigerator.

**P.S.** Steps 1, 3-6 should be operated on the ultra clean bench.

**Reagent**: bacterial fluid to be frozen, Sterile 50% glycerol, Sterile double distilled water, -80℃
Refrigerator

**Device**: Ultra clean bench, Pipette gun, Sterile pipette tips, Centrifuge, Centrifuge tube, 1.5 ml EP
tube

Procedure(for 20 ml bacterial fluid):

1. Take 20 ml of bacterial fluid whose OD600 reaches 0.6 in the suitable centrifuge tube.
2. Centrifuge to collect bacteria at 8000 g at 4℃ for 5 min.
3. Discard the supernatant.
4. Use 400 μl sterile 50% glycerol and 600 μl sterile double distilled water to resuspend in the 1.5 ml EP
   tube. Make the final concentration of glycerol reach 20%.
5. Label and put the EP tube into the -80℃ refrigerator.

**P.S.** Steps 1, 3-4 should be operated on the ultra clean bench.

Plasmid construction(Gibson assembly)

PCR

DNA PCR

**Reagent**: Double distilled water, 2xPhanta® Max Master Mix (Dye Plus) P525 or Golden
Mix(Green)(TSINGKE, production: TSE101), Template DNA(plasmid DNA), Forward primer(10μM), Reverse primer(10 μM),
Ice

**Device**: Pipette gun, Pipette tips, PCR tube, Icebox, PCR-Instrument, Centrifuge

Procedure:

* 1. Open the PCR-Instrument in advance.
* 2. Mix reagents on ice according to the table below in the PCR tube:

|  |  |
| --- | --- |
| **Components** | **Volume** |
| 2 × Phanta® Max Master Mix (Dye Plus) P525 or Golden Mix(Green) | 25 μl |
| Forward primer(10 μM) | 2 μl |
| Reverse primer(10 μM) | 2 μl |
| Template DNA(plasmid DNA) | xμl(10 pg-30 ng) |
| Double distilled water | up to 50 μl |

* 3. Mix the solution system evenly. Carry out rapid centrifugation if the droplets hang on the tube wall.
* 4. Put the PCR tube in the PCR-Instrument. Set the PCR program according to the table below.

|  |  |  |
| --- | --- | --- |
| **Stage/Number of Cycles** | **Temperature** | **Time** |
| Stage 1 / 1 95℃ | 30 s | |
| Stage 2 / 35 | 95℃ | 15 s |
| (Tm of primer-5)℃ | 15 s |
| 72℃ | 60 s/kb for Phanta® Max Master Mix |
| 15 s/kb for Golden Mix |
| Stage 3 / 1 72℃ | 5 min | |
| Stage 4 / 1 10℃ | forever | |

* 5. Take the tube out and put it in the icebox containing ice.

Colony PCR

**Reagent**: Double distilled water, 2xPhanta® Max Master Mix (Dye Plus) P525, Forward
primer(10 μM), Reverse primer(10 μM), Ice, Bacterial fluid to be used

**Device**: Pipette gun, Pipette tips, PCR tube, Icebox, PCR-Instrument, Centrifuge, Metal bath heater, 1.5
ml EP tube

Procedure:

* 1. Open the PCR-Instrument in advance. Set the metal bath heater to 95℃.
* 2. Add 100 μl bacterial fluid into a 1.5 ml EP tube. Put the tube on the metal bath heater for 10 minutes.
* 3. Mix reagents on ice according to the table below in the PCR tube:

|  |  |
| --- | --- |
| **Components** | **Volume** |
| 2 × Phanta® Max Master Mix (Dye Plus) P525 | 25 μl |
| Forward primer(10μM) | 2 μl |
| Reverse primer(10μM) | 2 μl |
| Heated bacterial fluid | 1-2 μl (according to the concentration of the bacteria) |
| Double distilled water | up to 50 μl |

* 3. Mix the solution system evenly. Carry out rapid centrifugation if the droplets hang on the tube wall.
* 4. Put the PCR tube in the PCR-Instrument. Set the PCR program according to the table below.

|  |  |  |
| --- | --- | --- |
| **Stage/Number of Cycles** | **Temperature** | **Time** |
| Stage 1 / 1 95℃ | 30 s | |
| Stage 2 / 35 | 95℃ | 15 s |
| (Tm of primer-5)℃ | 15 s |
| 72℃ | 60 s/kb |
| Stage 3 / 1 72℃ | 5 min | |
| Stage 4 / 1 10℃ | forever | |

* 5. Take the tube out and put it in the icebox containing ice.

Nucleic Acid Electrophoresis

Gel Making

**Reagent**: 1xTAE buffer, Agarose, 10000xNucleic acid dye

**Device**: Pipette gun, Pipette tips, Measuring cylinder, Electronic balance, Medicine spoon, Microwave
oven, Gel-making tank, Gel-making plate, Gel-making comb

Procedure(for a 1% nucleic acid electrophoresis gel):

1. Accurately weigh 0.3 g agarose and then add 30 ml of 1xTAE buffer.
2. Heat to boiling in the microwave oven at least 3 times to dissolve agarose completely.
3. Put a gel-making plate on the gel-making tank and put a gel-making comb on it.
4. Add 3 μl 10000xNucleic acid dye in the agarose solution when it's cooled to about 60℃.
5. Pour the agarose solution into the gel-making mold.
6. Wait about 20 minutes until the gel sets. Remove the comb.

Load sample and Electrophoresis

**Reagent**: Nucleic acid electrophoresis gel, DNA marker, PCR product, 1xTAE buffer

**Device**: Pipette gun, Pipette tips, Electrophoresis apparatus, Electrophoresis tank for nucleic acid

Procedure:

1. Add 1xTAE buffer into the electrophoresis tank for nucleic acid. Put the gel into the tank and make sure it
   can be submerged by TAE buffer.
2. Make sure the loading pore is close to the negative side.
3. Add 10 μl DNA marker into the loading pore. Add 50 μl PCR product into the loading pore. Record the position
   of the sample added.
4. Open the electrophoresis apparatus and connect it with the electrophoresis tank for nucleic acid.
5. Set the voltage to 100 V. Run 30 minutes or as appropriate.

**P.S.** The choice of DNA marker is based on the length of the PCR product.

Results acquisition

**Reagent**: Post-electrophoresis nucleic acid gel

**Device**: Gel imager(azure biosystems c150), Computer, Disposable gloves

Procedure:

1. Take out post-electrophoresis gel with disposable gloves on the hands.
2. Open the computer and gel imager. Open the software of the gel imager.
3. Put the gel in the imager. Set the exposure mode and capture the image of the gel.
4. Find the target bands and save the image.

Cutting Gel Recovery

Gel Recovery

**Reagent**: Post-electrophoresis nucleic acid gel, Gel recovery kit(FastPure® Gel DNA
Extraction Mini Kit DC301 from Vazynme), double distilled water

**Device**: UV Transilluminator, Scalpel, 1.5ml EP tube, Metal bath heater, Pipette gun, Pipette tips,
Electronic balance

Procedure:

1. Set the metal bath heater to 55℃ in advance.
2. Precisely cut the gel containing the target bands with a scalpel under the UV transilluminator.
3. Accurately weigh the pieces of gel. Put them into 1.5 ml EP tubes respectively.
4. According to the instructions in the kit recover the DNA fragment until the last step. Use double distilled
   water but not the buffer to dissolve the DNA fragment.

DNA Detection

**Reagent**: Gel recovery product, double distilled water

**Device**: NanoDrop one ultramicro ultraviolet spectrophotometer(Thermo SCIENTIFIC), Lens papers, Pipette
gun, Pipette tips

Procedure:

1. Open NanoDrop one in advance.
2. Select the "nucleic acid " tab and click the "double strain DNA" option on the home page of NanoDrop one.
3. Clean the instrument base with double distilled water. Wipe clean with lens papers.
4. Use 1 μl of double distilled water as a blank sample. After detecting, wipe clean with lens papers.
5. Add 1 μl gel recovery product to the base for detection. After detecting, record data and clean the
   instrument base with double distilled water. Wipe clean with lens papers.
6. Shut down the instrument after use.

Recombination

**Reagent**: Gel recovery product, double distilled water, Exnase II, 5xCE II buffer, Ice

**Device**: Pipette gun, Pipette tips, Icebox, PCR-Instrument, PCR tube

Procedure:

* 1. Open the PCR-instrument in advance.
* 2. Mix reagents on ice according to the table below in the PCR tube:

|  |  |
| --- | --- |
| **Components** | **Volume** |
| Linearization carrier | x μl (up to 0.03 pmol) |
| Insert Fragment | y μl (up to 0.06 pmol) |
| 5 × CE II buffer | 4 μl |
| Exnase II | 2 μl |
| Double distilled water | up to 20 μl |

* 3. Mix the solution system evenly. Carry out rapid centrifugation if the droplets hang on the tube wall.
* 4. Put the PCR tube in the PCR-Instrument. Set the PCR program according to the table below.

|  |  |  |
| --- | --- | --- |
| **Stage/Number of Cycles** | **Temperature** | **Time** |
| Stage 1 / 1 37℃ | 30 min | |
| Stage 2 / 1 10℃ | forever | |

* 5. Take the tube out and put it in the icebox containing ice.

Transformation and Coating

**Reagent**: Recombination product, DH5α competent *E. coli* cells, Ice, Resistant LB plate,
Non-resistant LB liquid medium

**Device**: Pipette gun, Sterile Pipette tips, Icebox, Metal bath heater, Ultra clean bench, Disposable
spreader, 1.5ml EP tube

Procedure:

1. Set the metal bath heater to 42℃ in advance.
2. Take DH5α competent *E. coli* cells and put them on the ice to thaw.
3. After thawing, add 10 μl recombination product into 100μl DH5α competent *E. coli* cells. Put it on ice
   for 30 minutes.
4. Heat shock the transformation tube for 90 seconds. Cool it on the ice for 3 minutes immediately after heat
   shock.
5. Add 600 μl non-resistant LB liquid medium in the transformation tube. Culture the transformed bacteria for
   45 minutes.
6. Use a disposable spreader to evenly coat 100 μl of the bacterial fluid on the resistant LB plate. Keep
   coating until no flowing liquid on the surface of the plate.
7. Seal the plate with parafilm. Label and put the plate in the thermostatic incubator.

**P.S.** The cultural time should not last too long, or the single colony can't be obtained.

Colony Selection and Amplificated Culture

**Reagent**: Antibiotic screening plate culturing transformed bacteria, Resistant LB liquid medium

**Device**: Pipette gun, Sterile Pipette tips, Ultra clean bench, Culture tubes

Procedure:

1. Use a sterile pipette tip to pick up the single colony on the plate.
2. Put the pipette tip stained with colony into the culture tube containing resistant LB liquid medium. Culture
   the bacterial fluid for about 12 hours.

Plasmid Extraction

Plasmid Extraction

**Reagent**: TIANprep Mini Plasmid Kit(TIANGEN® , product number: DP103-02) or
NucleoSpin® Plasmid, Mini kit for plasmid DNA(MACHEREY-NAGEL), double distilled water, Bacterial
fluid to be used

**Device**: 1.5 ml EP tube, Metal bath heater, Pipette gun, Pipette tips, Electronic balance, Centrifuge

Procedure:

1. Set metal bath heater to 55℃ in advance.
2. According to the instructions in the kit recover the DNA fragment until the last step. Use double distilled
   water but not the buffer to dissolve the plasmids.

DNA Detection

**Reagent**: Plasmid solution, double distilled water

**Device**: NanoDrop one ultramicro ultraviolet spectrophotometer(Thermo SCIENTIFIC), Lens papers, Pipette
gun, Pipette tips

Procedure:

1. Open NanoDrop one in advance.
2. Select the "nucleic acid " tab and click the "double strain DNA" option on the home page of NanoDrop one.
3. Clean the instrument base with double distilled water. Wipe clean with lens papers.
4. Use 1 μl of double distilled water as a blank sample. After detecting, wipe clean with lens papers.
5. Add 1 μl plasmid solution to the base for detection. After detecting, record data and clean the instrument
   base with double distilled water. Wipe clean with lens papers.
6. Shut down the instrument after use.

Experiments on *S. elongatus*

Measurement of Growth Curves

**Reagent**: *S. elongatus* bacterial fluid, Non-resistant BG11 liquid medium, Double distilled water

**Device**: Pipette gun, Sterile pipette tips, Ultra clean bench, 1.5 ml EP tube, NanoDrop One

Procedure:

1. 1:100 add the *S. elongatus* bacterial fluid to 200 ml BG11 liquid medium.
2. Remove 10 μl of bacterial fluid each time for measurement in the ultra clean bench. Measure and record the
   OD685 value.

Gene Manipulation by Natural Transformation

**Reagent**: Plasmid to be transformed, *S. elongatus* bacterial fluid, Non-resistant BG11 liquid
medium, Resistant BG11 plate

**Device**: Pipette gun, Sterile pipette tips, Ultra clean bench, Centrifuge, 1.5 ml EP tube, Thermostatic
light incubator, Disposable spreaders, Parafilm, Tinfoil, Thermostatic dark shaker

Procedure:

1. Take 1.5 ml of bacterial fluid whose OD685 reaches 0.5 in the 1.5 ml EP tube.
2. Centrifuge to collect bacteria at 6000 g for 10 min.
3. Discard the supernatant and use 750 μl of non-resistant BG11 liquid medium for resuspension. Centrifuge to
   collect bacteria at 6000 g for 10 min.
4. Repeat Step 3.
5. Discard the supernatant and use 250 μl of non-resistant BG11 liquid medium for resuspension.
6. Add plasmid solution into the tube. Make the concentration of DNA reach at least 1 ng/μl.
7. Wrap the 1.5 ml EP tube with tinfoil. Put it into the thermostatic dark shaker with 30℃ and 130 rpm for 24
   hours.
8. After 24 hours, use a disposable spreader to evenly coat 100 μl of the bacterial fluid on the resistant BG11
   plate. Keep coating until no flowing liquid on the surface of the plate.
9. Seal the plate with parafilm. Label and put the plate in the thermostatic light incubator. Culture for 5
   days or until the single colony occurs. These single colonies are transformants.

**P.S.** Steps 1, 3-6, 8 should be operated on the ultra clean bench.

Induced expression

**Reagent**: *S. elongatus* bacterial fluid to be induced, 1M IPTG solution, Resistant BG11 liquid
medium

**Device**: Pipette gun, Sterile pipette tips, Ultra clean bench, Centrifuge, Centrifuge tube, Thermostatic
shaker with external lamp tube

Procedure(for 5 ml bacterial fluid):

1. Take 4.995 ml of bacterial fluid whose OD685 reaches 0.5 in the centrifuge tube.
2. Centrifuge to collect bacteria at 6000 g for 10 min.
3. Discard the supernatant and use 1ml of non-resistant BG11 liquid medium for resuspension. Centrifuge to
   collect bacteria at 6000 g for 10 min.
4. Discard the supernatant and use 4.995 ml of resistant BG11 liquid medium for resuspension.
5. Add 5 μl of 1 M IPTG solution into the bacterial fluid. Mix them evenly.
6. Label and put it into the thermostatic shaker with an external lamp tube with 30℃ and 130 rpm.

**P.S.** Steps 1, 3-5 should be operated on the ultra clean bench.

Experiment on The Production of Sucrose

**Reagent**: Wild-type *S. elongatus* bacterial fluid, *S. elongatus* bacterial fluid transformed
with CscB gene, Non-resistant BG11 liquid medium, Resistant BG11 liquid medium, 1 M IPTG solution, 1 M NaCl
solution, Plant Sucrose Content Assay Kit(boxbio, product number: AKPL006M)

**Device**: Pipette gun, Sterile pipette tips, Ultra clean bench, Culture tube, Thermostatic shaker with
external lamp tube, Centrifuge, Centrifuge tube

Procedure:

* 1. Take 60 ml of wild-type *S. elongatus* bacterial fluid whose OD685 reaches 0.5 in the
  centrifuge tube. Take 60 ml of *S. elongatus* bacterial fluid transformed with CscB gene whose
  OD685 reaches 0.5 in the centrifuge tube.
* 2. Centrifuge each group to collect bacteria at 6000 g for 10 min.
* 3. Discard the supernatant and use the non-resistant BG11 liquid medium for resuspension of both groups.
  Centrifuge to collect bacteria at 6000 g for 10 min.
* 4. Discard the supernatant. Use 60 ml of non-resistant BG11 liquid medium for resuspension of wild-type
  *S. elongatus*. Use 60 ml of resistant BG11 liquid medium for resuspension of *S. elongatus*
  transformed with CscB gene.
* 5. Mix reagents according to the table below in the culture tube: (C represents the control groups; E
  represents the experiment groups)

|  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  | **Group C-1** | **Group C-2** | **Group C-3** | **Group C-4** | **Group E-1** | **Group E-2** | **Group E-3** | **Group E-4** |
| **Wild-type *S. elongatus* bacterial fluid (μl)** | 5000 | 4995 | 4500 | 4495 | 0 | 0 | 0 | 0 |
| ***S. elongatus* bacterial fluid transformed with cscB gene (μl)** | 0 | 0 | 0 | 0 | 5000 | 4995 | 4500 | 4495 |
| **1M IPTG solution (μl)** | 0 | 5 | 0 | 5 | 0 | 5 | 0 | 5 |
| **1M NaCl solution (μl)** | 0 | 0 | 500 | 500 | 0 | 0 | 500 | 500 |
| **Final volume of fluid (ml)** | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
| **Number of parallel groups** | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 |

* 6. Label and put them into the thermostatic shaker with an external lamp tube with 30℃ and 130 rpm. Start
  timing at the same time.
* 7. Measure and record OD685 value at 0 hours, 24 hours, 48 hours, and 72 hours.
* 8. Take 100 μl bacterial fluid from each tube at 24 hours, 48 hours, and 72 hours. Centrifuge to obtain
  supernatant at 6000 g for 10 min.
* 9. Measure and record the concentration of sucrose in the supernatant with the assay kit.

**P.S.** Steps 1, 3-5, 8, and all sampling operation should be operated on the ultra clean bench.

Experiments on *E. coli*

Preparation of Seeds

**Reagent**: *E. coli* bacterial fluid transformed with recombinant plasmid, *E. coli* bacterial
fluid transformed with empty vector, Resistant LB liquid medium, M9 minimal liquid medium with
sucrose

**Device**: Ultra clean bench, Pipette gun, Sterile pipette tips, NanoDrop One

Procedure:

1. Culture engineering *E. coli* transformed with recombinant plasmid and engineering *E. coli*
   transformed with empty vector in 3 ml resistant LB liquid medium until OD600 reaches 0.6.
2. Store them at 4℃ as seeds for the following steps.

Sucrose Utilizing

**Reagent**: *E. coli* seeds with recombinant plasmid, *E. coli* seeds with empty vector,
Resistant LB liquid medium, M9 minimal liquid medium with sucrose

**Device**: Ultra clean bench, Pipette gun,
Sterile pipette tips, NanoDrop One

Procedure:

1. 1:100 add the seeds required for the certain experiment to 100 ml M9 minimal liquid medium with sucrose
   respectively. Add antibiotics corresponding to the resistance they have.
2. Culture these groups of *E. coli*. Measure and record their OD600 every 2 hours. Take out 80
   μl of liquid on the ultra clean bench for the test each time. If OD600 is higher than 1.5, measure
   again after dilution until its OD600 is lower than 1.5.

**P.S.** The plasmids required for our experiments contain pUC-Empty, pUC-J23101-SacC, pUC-J23107-SacC, and
pUC-J23109-SacC.

Starvation Response

**Reagent**: *E. coli* seeds with recombinant plasmid, *E. coli* seeds with empty vector, Resistant
LB liquid medium, M9 minimal liquid medium with different glucose concentration

**Device**: Ultra clean bench, Pipette gun, Sterile pipette tips, NanoDrop 100, Black 96-well plate,
Transparent 96-well plate

Procedure:

1. Preparation of M9 minimal medium: In 1L M9 minimal medium 33.9g Na2HPO4, 15g KH2PO4, 2.5g NaCl, 5g NH4Cl,
   0.36g MgSO4, glucose with a concentration gradient is supplied as the sole carbon source.
2. Dilute the *E. coli* cultures that are stored at 4 degrees 1:100 into 3ml LB medium and culture
   overnight.
3. Then the cultures were diluted 1:100 into 3mL LB medium and cultured for about 3 hours. Use these
   cultures(OD600 0.6~0.8) as seeds.
4. Dilute the seeds 1:100 to 600ul M9 minimal medium and culture for 6 hours. Add 200ul into each well of
   96-well plate. Measure OD600 and fluorescence intensity of each sample with a multimode plate reader.

Quorum Sensing

**Reagent**: *E. coli* seeds transformed with [BBa\_K4115039](http://parts.igem.org/Part%3ABBa_K4115039), *E. coli* seeds transformed with [BBa\_K4115040](http://parts.igem.org/Part%3ABBa_K4115040), Resistant LB liquid medium, Autoinducer(3O C6
HSL) solution

**Device**: Ultra clean bench, Pipette gun, Sterile pipette tips, NanoDrop one, Transparent
96-well plate, 1.5 ml EP tube, Black bottom 96-well plate, Centrifuge, Centrifuge tube

Procedure:

**Signal Sender**

* 1. Dilute the *E. coli* seeds transformed with [BBa\_K4115039](http://parts.igem.org/Part%3ABBa_K4115039) 1:100 into 3ml LB liquid medium and culture
  overnight.
* 2. Dilute the overnight cultures 1:100 into 3ml LB liquid medium and culture for 3h. Measure OD600 with
  Nanodrop One.
* 3. Dilute the culture with LB liquid medium until OD600 is about 0.1. Add 600 μl diluted cultures
  to each 1.5ml EP tube. Add 0.6 μl autoinducer solution to each tube. Make the final concentrations of the
  autoinducer reach the range from 10-4 to 10-14 M. Then culture them.
* 4. After culturing for 3 hours, add 200 μl to each well of the black bottom 96-well plate for fluorescence
  intensity testing. Use an excitation wavelength of 485nm and an emission wavelength of 535nm. Record the data.
  Record the data.
* 5. Add 200 μl to each well of the transparent 96-well plate for OD600 measurement.

**Signal Receiver**

* 1. Dilute the *E. coli* seeds transformed with [BBa\_K4115040](http://parts.igem.org/Part%3ABBa_K4115040) 1:100 into LB liquid medium and culture
  overnight. Dilute the *E. coli* seeds transformed with [BBa\_K4115039](http://parts.igem.org/Part%3ABBa_K4115039) 1:100 into 3ml LB liquid medium and culture
  overnight.
* 2. Centrifuge the overnight *E. coli* bacterial fluid transformed with [BBa\_K4115040](http://parts.igem.org/Part%3ABBa_K4115040) (express LuxI constitutively) at 12000 rpm
  for 1 min. Take out the supernatants for use.
* 3. Dilute the overnight *E. coli* bacterial fluid transformed with [BBa\_K4115039](http://parts.igem.org/Part%3ABBa_K4115039) until OD600 is about 0.1.
* 4. Set 3 groups as follows:

|  |  |  |  |
| --- | --- | --- | --- |
| **Group name** | Blank | Autoinducer | Signal sender |
| **Medium** | LB medium | LB medium with 10-7 autoinducer | Supernatant from Step 2 |

1:1000 add the 10^-3 M autoinducer into the diluted cultures in order to set the autoinducer group. Centrifuge
the diluted cultures at 3500 rpm for 10 min and then resuspend the cells with the supernatant in Step 2 in order
to set the signal sender group.

* 5. Culture all these three groups for 3h.
* 6. After culturing, add 200 μl of bacterial fluid of each group to each well of the black bottom 96-well
  plate for fluorescence intensity testing from each group. Use an excitation wavelength of 485nm and an
  emission wavelength of 535nm.
* 7. Add 200 μl of bacterial fluid of each group to each well of the transparent 96-well plate for
  OD600 measurement.

Experiments on *A. caulinodans*

Measurement of Growth Curves

In TY Medium

**Reagent**: Initial bacterial fluid, Ice, Resistant TY liquid medium

**Device**: Ultra clean bench, Culture tubes, Pipette gun, Sterile pipette tips, NanoDrop One

Procedure:

1. Seed initial cultures at a ratio of 1:100 into 100 ml of TY liquid medium containing 100 μg/ml ampicillin
   and culture.
2. Measure and record the OD600 value of the culture medium every 4 hours.

In CoBG11-Acetate Medium

**Reagent**: Initial bacterial fluid, BG11 medium powder(hopebio, product number: HB8793), NH4Cl,
NaCl, HEPPS, Milli-Q water, Acetate, Sucrose

**Device**: Ultra clean bench, Measuring cylinder, Electronic balance, Medicine spoon, BeyoGold Syringe
Filter (0.22μm/3mm, PES, Sterile)

Procedure:

1. Dissolve 11.7 g BG11 powder, 106 mM sodium chloride, 4 mM ammonium chloride, 25 mM HEPPS and 10 mM acetate
   into 1 L Milli-Q water. Adjust the pH to 7.5. Mark this solution as CoBG11-acetate.
2. Dissolve 11.7 g BG11 powder, 106 mM sodium chloride, 4 mM ammonium chloride, 25 mM HEPPS and 20 g sucrose
   into 1 L Milli-Q water. Adjust the pH to 7.5. Mark this solution as CoBG11-sucrose.
3. Use the filter to filter the both solution.
4. Seed initial cultures at a ratio of 1:100 into 100 ml CoBG11-acetate or CoBG11-sucrose liquid medium in a
   shaker at 37℃, 180 rpm.
5. Measure and record the OD600 value of the culture medium every day.

Electrotransformation

Preparation

**Reagent**: Recovered bacterial fluid, Ice, Resistant TY liquid medium, Sterile double distilled water,
Sterile 10% glycerol

**Device**: Ultra clean bench, Icebox, Culture tubes, Pipette gun, Sterile pipette tips, Centrifuge,
Centrifuge tube, Refrigerator, 1.5 ml EP tube

Procedure:

1. Seed initial cultures at a ratio of 1:100 into 100 ml of TY liquid medium containing 100 μg/ml ampicillin
   and culture until OD600 = 0.4 ~ 0.6.
2. Pre-cool sterile double distilled water and sterile 10% glycerol.
3. Place the cultures on ice for 30 min, then centrifuge the culture medium at 6000 rpm for 10 min to remove
   the supernatant and resuspend in 35 ml sterile pre-cold water.
4. Centrifuge the culture medium again at 6000 rpm for 10 min to remove the supernatant and resuspend in 20 ml
   sterile pre-cold water.
5. Repeat the last step but resuspend the bacteria with 2.5 ml sterile pre-cold 10% glycerol instead of 20 ml
   water.
6. Divide the glycerol bacteria into sterile 1.5 ml EP tubes (150 μl each), and store at -80℃.

Electrotransformation

**Reagent**: Prepared glycerol bacterial, Ice, Resistant TY liquid medium, Sterile double distilled water,
Absolute ethanol, Plasmid solution to be transformed

**Device**: Ultra clean bench, Icebox, Culture tubes, Pipette gun, Sterile pipette tips, Centrifuge,
Centrifuge tube, Refrigerator, 1.5 ml EP tube, electroporation cuvettes, BIO-RAD MicroPulser

Procedure:

1. re-cool sterile double distilled water and absolute ethanol.
2. Wash the 2 mm electroporation cuvettes with pre-cold sterile water and pre-cold absolute ethanol
   successively.
3. Melt the electrocompetent cells on ice. Add 10 μl plasmid solution into cells, and leave it on ice for 30
   min.
4. Transfer cells into a cuvette and avoid bubbles in the liquid.
5. Use *BIO-RAD MicroPulser* to electroporate at 2500V for 5ms.
6. Transfer cells back to 1.5 ml EP tube, add 800 μl TY liquid medium and culture for 7~8 hours.
7. Centrifuge the culture medium at 4000 rpm for 4 min, remove 700 μl supernatant and resuspend the cell with
   the remaining liquid.
8. Perform the spread plate method for the concentrated cell liquid on TY solid medium containing the correct
   antibiotics and culture for 1 day.

Extraction of Genomic DNA

**Reagent**: Bacterial plate, TIANamp Bacteria DNA Kit(TIANGEN)

**Device**: Pipette gun, Pipette tips, Centrifuge, Centrifuge tube, 1.5 ml EP tube

Procedure:

1. Inoculate single colonies into 4 ml of TY liquid medium containing 100 μg/ml ampicillin and 100 μg/ml
   bleomycin and culture for 1 day.
2. Extract the genomic DNA of *A. caulinodans* by the TIANamp Bacteria DNA Kit.

Co-Culture

Experiment on Culturing *E. coli* With the Supernatant of *S. elongatus*

**Reagent**: *S. elongatus* bacterial fluid transformed with CscB gene, 1 M IPTG solution, 1 M NaCl
solution, Plant Sucrose Content Assay Kit(boxbio, product number: AKPL006M), *E. coli* bacterial fluid
transformed with SacC gene, Resistant LB liquid medium, Non-resistant CoBG11 liquid medium, NaCl-free
non-resistant CoBG11 liquid medium, 20% Sucrose solution

**Device**: Pipette gun, Sterile pipette tips, Refrigerator, Ultra clean bench, Culture tube, Thermostatic
shaker with external lamp tube, Centrifuge, Centrifuge tube, Thermostatic shakerProcedure:

* 1. Take two portions of 50 ml of *S. elongatus* bacterial fluid transformed with CscB gene, whose
  OD685 reaches 0.5 in the centrifuge tube. Mark these two portions as group 1 and group 2
  respectively.
* 2. Centrifuge each group to collect bacteria at 6000 g for 10 min.
* 3. Discard the supernatant. Use the non-resistant CoBG11 liquid medium for resuspension of group 1. Use the
  NaCl-free non-resistant CoBG11 liquid medium for resuspension of group 2. Centrifuge each group to collect
  bacteria at 6000 g for 10 min.
* 4. Discard the supernatant. Use 50 ml of non-resistant CoBG11 liquid medium that is already added 50 μl of 1
  M IPTG solution for resuspension of group 1. Use 50 ml of NaCl-free non-resistant CoBG11 liquid medium for
  resuspension of group2.
* 5. Culture both two groups for 84 hours. Measure and record OD685 value. The data of group 1 is
  recorded as OD1 and OD2 for group 2.
* 6. Centrifuge each group to collect supernatant at 6000 g for 10 min. Mark the supernatant of group 1 as the
  supernatant+ and that of group 2 as the supernatant-.
* 7. Dilute the supernatant of the group with large OD value in a ratio of their OD value(higher than 1).
* 8. Add 1 M NaCl solution into supernatant- and make the final concentration of NaCl reach 106
  mM(for instance, add 4.76 ml of 1 M NaCl solution into 40 ml supernatant-).
* 9. Measure and record the concentration of sucrose in the both two groups of supernatant with the assay kit.
* 10. 1:100 add *E. coli* bacterial fluid transformed with SacC gene into 50 ml resistant LB liquid
  medium. Culture until the OD600 reaches 0.5.
* 11. Add 20% sucrose solution into the non-resistant CoBG11 liquid medium and make the concentrate of sucrose
  reach the higher value measured in Step 9.
* 12. Prepare four solutions for the following steps according to the table below.

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| **Label** | Solution 1 | Solution 2 | Solution 3 | Solution 4 |
| **Component** | Non-resistant CoBG11 | Non-resistant CoBG11 with sucrose from Step11 | Supernatant- | Supernatant+ |
| **Volume (ml)** | 40 | | | |

* 13. The volume of *E. coli* bacterial fluid to be used mainly depends on its OD600. The
  following steps take OD600=0.5 as an example.
* 14. Take four portions of 10.5 ml of *E.coli* bacterial fluid in centrifuge tube. Centrifuge all
  portions to collect bacteria at 4000 g for 10 min.
* 15. Discard the supernatant and use the non-resistant CoBG11 liquid medium for resuspension of all portions.
  Centrifuge all portions to collect bacteria at 4000 g for 10 min. Mark four portions as group 1 - 4.
* 16. Suspend group 1 - 4 of *E. coli* with 21 ml of solution 1 - 4 respectively and obtain 100%
  bacterial fluid. Set the control groups and experiment groups according to the following table.

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| 100% bacterial fluid | | | | |
| **Group** | 1 | 2 | 3 | 4 |
| **Component** | Solution 1 | Solution 2 | Solution 3 | Solution 4 |
| **Theoretical OD600 value** | 0.25 | | | |
| **Volume per tube (ml)** | 4 | | | |
| **Number of tubes** | 3 | | | |

* 17. Dilute the remaining 9 ml of 100% bacterial fluid with 9 ml of the corresponding solution and obtain 50%
  bacterial fluid. Set the control groups and experiment groups according to the following table.

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| 50% bacterial fluid | | | | |
| **Group** | 1 | 2 | 3 | 4 |
| **Component** | Solution 1 | Solution 2 | Solution 3 | Solution 4 |
| **Theoretical OD600 value** | 0.13 | | | |
| **Volume per tube (ml)** | 4 | | | |
| **Number of tubes** | 3 | | | |

* 18. Dilute the remaining 6 ml of 50% bacterial fluid with 6 ml of the corresponding solution and obtain 25%
  bacterial fluid. Set the control groups and experiment groups according to the following table.

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| 25% bacterial fluid | | | | |
| **Group** | 1 | 2 | 3 | 4 |
| **Component** | Solution 1 | Solution 2 | Solution 3 | Solution 4 |
| **Theoretical OD600 value** | 0.06 | | | |
| **Volume per tube (ml)** | 4 | | | |
| **Number of tubes** | 3 | | | |

* 19. Measure and record OD600 as the value of 0 hours. Label and put them into the thermostatic
  shaker to culture. Start timing at the same time.
* 20. Measure and record OD600 every 3 hours until culturing them for 48 hours.

Measurement of Growth Curve of the Three

**Reagent**: Initial *E .coli* bacterial fluid, Initial *S .elongatus* bacterial fluid, Initial
*A .caulinodans* bacterial fluid, CoBG11 liquid medium, Sucrose

**Device**: Pipette gun, Sterile Pipette tips, Centrifuge, Centrifuge tube, 1.5 ml EP tube, NanaDrop One,
BeyoGold Syringe Filter (0.22μm/3mm, PES, Sterile), Thermostatic shakerProcedure:

1. Add 20 g of sucrose into the CoBG11 liquid medium to obtain CoBG11-sucrose liquid medium. Sterilize by the
   filter.
2. Seed initial cultures at a ratio of 1:100 for *E. coli* and *A. caulinodans*, and at a ratio of
   1:20 for *S. elongatus* into 100 ml CoBG11-sucrose liquid medium in a thermostatic shaker(for *E.
   coli*, at 37℃, 220 rpm; for *A. caulinodans*, at 37℃, 180 rpm; for *S. elongatus*, at 30℃, 130
   rpm with lamp tube).
3. Measure and record the OD value of each culture medium every 4h (*E. coli*), 6h(*A. caulinodans*),
   or 12h (*S. elongatus*).

Immobilization of *E. coli* and *S. elongatus*

**Reagent**: Initial *E .coli* bacterial fluid, Initial *S .elongatus* bacterial fluid, Sodium
alginate powder, Calcium chloride, CoBG11-sucrose liquid medium and CoBG11-acetate liquid medium(see
'Experiments on *A. caulinodans*'-'Measurement of Growth Curves'), Physiological saline, Milli-Q water

**Device**: Pipette gun, Sterile Pipette tips, Centrifuge, Centrifuge tube, 1.5 ml EP tube, NanaDrop One,
BeyoGold Syringe Filter (0.22μm/3mm, PES, Sterile), Thermostatic shaker, Microscope, Coverslip, Fluorescence
microscope

Preparation of Sodium Alginate Solution

Procedure:

1. Add 2 g sodium alginate powder to 100 ml hot water, heat and stir until completely dissolved.
2. Leave sealed for several hours until the bubbles all disappear.

Preparation of Calcium Chloride Solution

Procedure:

1. Add 2.22 g calcium chloride to 100 ml Milli-Q water. Make the concentration of the calcium chloride reach
   0.2 M.
2. Use the filter to filter the solution.

Immobilization of Microorganisms

Procedure:

1. Centrifuge the microorganism fluid at 4000 rpm for 15 min. Remove the supernatant and weigh the wet weight.
2. For every 1 g cell, resuspend in 1 ml physiological saline, add 20 ml sodium alginate solution and stir
   until well combined.
3. Transfer the alginate-cell mixture into a small syringe (needle removed) and drips naturally into 20 ml 0.2
   M calcium chloride solution. Meanwhile, stir with an electromagnetic stirrer.
4. Soak for 5 hours, then transfer the immobilized cells into CoBG11-sucrose medium (for *E. coli* and
   *S. elongatus*) or CoBG11-acetate medium (for *A. caulinodans*), culture in a shaker (for *E.
   coli*, at 37℃, 220 rpm; for *A. caulinodans*, at 37℃, 180 rpm; for *S. elongatus*, at 30℃, 130
   rpm).

Lysis of Immobilized Cells and Measurement

Procedure:

1. Transfer the immobilized cells into 1.5 ml EP tubes and weigh wet weight.
2. For every 1 g immobilized cell, add 1 ml 0.2 M phosphate buffer (pH=7.5).
3. Shake violently for 15 min until all solids dissolve.
4. Measure the OD600 (for *E. coli* and *A. caulinodans*) or OD685 (for *S.
   elongatus*) value of the lysate.
5. Perform the above operations once a day for about a week.

Fluorescence Image Analysis.

Procedure:

1. Drop 10 μl lysate onto the microslide and cover the coverslip.
2. Place the sample under a fluorescence microscope and obtain the image.
3. Perform image analysis to get the average fluorescence intensity.

Co-culture of Immobilized Microorganisms

**Reagent**: Initial *E .coli* bacterial fluid, Initial *S .elongatus* bacterial fluid, Initial
*A .caulinodans* bacterial fluid, Sodium alginate powder, Calcium chloride, CoBG11-sucrose liquid medium
and CoBG11-acetate liquid medium(see Experiments on *A. caulinodans*-Measurement of Growth Curves),
Physiological saline, Milli-Q water

**Device**: Pipette gun, Sterile Pipette tips, Centrifuge, Centrifuge tube, 1.5 ml EP tube, NanaDrop One,
BeyoGold Syringe Filter (0.22μm/3mm, PES, Sterile), Thermostatic shaker, Microscope, Coverslip, Fluorescence
microscope

Preparation of CoBG11-PLUS Medium

Procedure:

1. Dissolve 11.7g BG11 powder, 106mM sodium chloride, 4mM ammonium chloride, 25mM HEPPS, 20g sucrose, and 10mM
   acetate into 1 l Milli-Q water. Adjust the pH to 7.5.
2. Use the filter to filter the solution.

Immobilization of Microorganisms

Procedure:

1. Centrifuge the microorganism fluid at 4000 rpm for 15 min. Remove the supernatant and weigh the wet weight.
2. For every 1 g cell, resuspend in 1 ml physiological saline, add 20 ml sodium alginate solution and stir
   until well combined.
3. Transfer the alginate-cell mixture into a small syringe (needle removed) and drips naturally into 20 ml 0.2
   M calcium chloride solution. Meanwhile, stir with an electromagnetic stirrer.
4. Soak for 5 hours, then transfer the immobilized cells into CoBG11-PLUS medium and culture in a light shaker
   at 30℃, 130 rpm.

Lysis of Immobilized Cells and Measurement

Procedure:

1. Transfer the immobilized cells into 1.5 ml EP tubes and weigh wet weight.
2. For every 1 g immobilized cell, add 1 ml 0.2 M phosphate buffer (pH=7.5).
3. Shake violently for 15 min until all solids dissolve.
4. Measure the OD600 (for *E. coli* and *A. caulinodans*) or OD685 (for *S.
   elongatus*) value of the lysate.
5. Perform the above operations once a day for about a week.

Intercellular Communication in Immobilized *E .coli* Through LuxR System

**Reagent**: *E .coli* bacterial fluid carries LuxI gene, *E .coli* bacterial fluid carries
LuxR-Plux-sfGFP sequence, CoBG11-sucrose liquid medium(see 'Experiments on *A. caulinodans*'-'Measurement
of Growth Curves'), Physiological saline, Sodium alginate solution

**Device**: Pipette gun, Sterile Pipette tips, Centrifuge, Centrifuge tube, 1.5 ml EP tube, NanaDrop One,
BeyoGold Syringe Filter (0.22μm/3mm, PES, Sterile), Thermostatic shakerProcedure:

1. Centrifuge the microorganisms culture at 4000 rpm for 15 min, remove the supernatant and weigh wet weight.
2. For every 1 g cell, resuspend in 1 ml physiological saline, add 20 ml sodium alginate solution and stir
   until well combined.
3. Transfer the alginate-cell mixture into a small syringe (needle removed) and drips naturally into 20 ml 0.2
   M calcium chloride solution. Meanwhile, stir with an electromagnetic stirrer.
4. Soak for 5 hours, then transfer the immobilized cells into CoBG11-sucrose medium and culture in a shaker at
   37℃, 100 rpm.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/4115/wiki/logowhite.svg)
![](https://static.igem.wiki/teams/4115/wiki/logowhite.svg)

Results

* [Overview](#1)
* [E. coli](#2)
* [S. elongatus](#3)
* [A. caulinodans](#4)
* [Immobilization and
  Co-Culture](#5)
* [References](#6)

Overview

Our ternary microbial symbiosis system is consist of three chassis: *Escherichia coli*, *Synechococcus
elongatus* , and *Azotobacter caulinodans*. *S. elongatus* fix CO2 through
photosynthesis and secretes the product, sucrose as the carbon source for heterotrophic chassis. *A.
caulinodans* convert the N2 in Mars atmosphere into the universal nitrogen source,
NH4+. *E. coli* plays the role of production chassis.

To achieve high stability and production efficiency, two strategies are applied to our symbiosis system. First,
we hope to build up an effective **nutrient circulation** among the above three microorganisms. Second, we
construct **intercellular genetic circuit** feedback to regulate the nutrient flux in our system. In the
genetic circuit, starvation promoters in *E. coli* are sensors for nutrient signals. The *lux* quorum
sensing system undertakes the function of transmitting intercellular signals and acts on *S. elongatus* and
*A. caulinodans* to regulate nutrient fixation efficiency.

For implementation, suitable microbial fermentation equipment and pattern for our system are particularly
important. We proposed a novel fermentation model named **Separate-Immobilized Fermentation**. To see more
details of the designs of our project, please go to our [Description](https://2022.igem.wiki/shanghaitech-china/description) page.

We will present the experimental results on nutrient circulation and intercellular genetic circuit on the three
chassis in order, and present immobilized culturing-related data in the fourth part.

*E. coli*

Nutrient circulation

Sucrose utilization

The carbon source produced by *S. elongatus* is provided to *E. coli* in the form of sucrose.
However, some previous papers have reported that wild-type **E. coli** K12 strains can't utilize the
sucrose to grow and produce very well[1]. To improve the sucrose utilization efficiency, we have
expressed a fructofuranosidase (SacC) from *Mannheimia succiniciproducens*. SacC will be secreted to
intercellular space and hydrolyze sucrose into glucose and fructose. Then these two monosaccharides can be
uptaken through the PEP-dependent phosphotransferase system efficiently (Figure 1).

![results-SacC_mechanism](https://static.igem.wiki/teams/4115/wiki/results-sacc-mechanism.png)

**Figure 1.** The mechanism of SacC in enhancing sucrose utilizing.Sucrose will be hydrolyzed by SacC into
two glucose and fructose, which will be uptaken by the two PTSs

A series of constructs expressing SacC with different transcriptional activity was constructed and then
transformed into *E. coli* DH5α (Figure2). Based on the activities of Anderson promoters have been well
quantified, the
order of expression level of these four constructs is expected as follows: J23101-SacC > J23107-SacC >
J23109-SacC > Empty.

![](https://static.igem.wiki/teams/4115/wiki/results-sacc-constructs.png)

**Figure 2.** Four constructs expressing SacC at different levels.The expected expression levels are
shown on the right.

Then we cultured all these four strains in an M9 minimal medium which contains sucrose as the sole carbon
source. Their growth is quantified by measuring optical density at 600 nm (OD600). All of the groups
expressing SacC grow faster than Empty in the first 14 hours. Furthermore, compared with J23109-SacC, the SacC
high expression groups J23101-SacC and J23107-SacC showed higher growth rates, along with the dose-dependent
phenomenon (Figure 3A). After culturing for 10 hours, we take out the culture medium to test the remaining
sucrose. The residual sucrose concentrations of culture medium in the SacC-positive groups were significantly
lower than that of Empty (Figure 3B). The higher the SacC expression level, the lower the remaining sucrose
concentration. These demonstrate that the appropriate expression level of SacC can help *E. coli* use
sucrose as the sole carbon source to grow.

![](https://static.igem.wiki/teams/4115/wiki/results-sacc-growth-curve.png)

![](https://static.igem.wiki/teams/4115/wiki/results-sacc-remaining-sucrose.png)

**Figure 3.** Sucrose utilization profiles of *E. coli* strains in the medium containing sucrose as a
sole carbon source. (A) Growth curves of four strains. (B) Remaining sucrose concentrations in culture medium
after 10 hours. Blank medium: the M9 minimal medium without adding *E.coli* was kept at the same
condition as other groups for 10 hours.

Unexpectedly, Empty reaches a higher OD600 at the stationary phase. We hypothesize that this
difference may be caused by a metabolism pathway difference between SacC-positive groups and Empty. After 14
hours, SacC-positive groups and Empty may accumulate different by-products. For example, fast-growing *E.
coli* will produce more acetic acid, a common phenomenon called overflow metabolism[18]. To test
this hypothesis, we measure the pH value of the medium after culturing for 48 hours. pH value decrease was found
in SacC high expression groups, which indicates that they produce more organic acids (Figure 4). Previous
research has proved that organic acids like acetate can inhibit the growth of *E. coli*, which may cause
the lower stationary phase of SacC-positive groups[19]. Moreover, acetate can not be utilized as
efficiently as glucose and fructose, so acetate production is a waste of carbon source. This might be another
reason for the lower stationary phase.

![](https://static.igem.wiki/teams/4115/wiki/results-ph-8-11.jpg)

**Figure 4.** pH value of medium after culturing for 48 hours. From left to right: Empty, J23101-SacC,
J23107-SacC, J23109-SacC

Intercellular genetic circuit

Considering *E. coli* is the production chassis in our system, we set *E. coli* as the core and
initiator of the genetic circuit. Our engineered *E. coli* is given the ability of **nutrient starvation
response**, and then 'tell' the nutrition situation to the nitrogen fixation bacteria and cyanobacteria
through a **quorum sensing system**.

Nutrient starvation response

The stationary phase sigma factor RpoS and the signal molecule cAMP are the major elements in the regulation of
transcription under carbon source starvation conditions.[2, 3] These two regulators are involved in
two pathways of starvation response, respectively (Figure 5). RpoS is an alternative sigma factor. As with other
sigma factors, RpoS binds to the core RNA polymerase and controls the expression of a specific but large set of
genes related to stress resistance. Under the conditions of nutrient deprivation or other stresses, the
intracellular abundance of RpoS will increase, which will accelerate the transcription initiation of the
RpoS-dependent genes (Figure 5A). The other starvation response pathway uses a cAMP receptor protein (CRP) as a
regulator and the sigma factor RpoD for transcription initiation. Under carbon source starvation conditions, the
intracellular cAMP concentration will increase and further activate CRP. Then CRP binds to a CRP-binding
sequence located upstream of the transcription start site and activates this CRP-dependent promoter (Figure 5B).

![](https://static.igem.wiki/teams/4115/wiki/results-starvation-mechanism.png)

**Figure 5.** Two pathways of starvation response. (A) RpoS-dependent (B) CRP-dependent

To achieve the nutrient starvation response, we first selected three endogenous carbon starvation promoters as
the sensors: PyciG ([BBa\_K4115018](http://parts.igem.org/Part%3ABBa_K4115018)), PcstA ([BBa\_K4115003](http://parts.igem.org/Part%3ABBa_K4115003) or [BBa\_K118011](http://parts.igem.org/Part%3ABBa_K118011)), and PcsiE ([BBa\_K4115016](http://parts.igem.org/Part%3ABBa_K4115016)). These promoters use different
transcriptional factors and sigma factors (Table 1).

|  |  |  |
| --- | --- | --- |
|  | **Trancriptional factors** | **Sigma factors** |
| PyciG[4] | Unknown | RpoS |
| PcstA[5] | cAMP receptor protein (CRP); factor for inversion stimulation (Fis) | RpoD |
| PcsiE[6] | cAMP receptor protein (CRP) | RpoD; RpoS |

**Table 1.** Transcriptional factors and sigma factors used by PyciG, PcstA and PcsiE.

To check if the selected promoters can be activated by nutrient limitation (starvation), we constructed report
genes as Figure 6A. *E. coli* transformed with such report genes are cultured in an M9 medium containing
glucose with different concentrations as the sole carbon source. After 6 hours, the cultures are taken for
measuring fluorescence intensity (FI) and OD600. **The fluorescence intensities normalized by
OD600 indicate the relative promoter activities.**
After communicating with our partnership
team BUCT\_China, we decide to add an **LVA degradation tag** on the C-terminal of sfGFP. (To see more
collaborations with BUCT\_China, you can go to our [partnership](https://2022.igem.wiki/shanghaitech-china/partnership) page.) sfGFP has a very long
half-life in the cytoplasm, which makes it not suitable for indicating some immediate change in gene expression.
LVA tag can reduce the half-life. So in principle, using sfGFP with LVA tag as the reporter can more
realistically reflect changes in promoter activity (Figure 6B).

![](https://static.igem.wiki/teams/4115/wiki/results-starvation-promoter.png)

**Figure 6.** sfGFP (A) or sfGFP with LVA (B) are used as a reporter to indicate starvation promoter
activity.

The following data in Figure 7 demonstrates that all three selected promoters have a higher promoter activity
under the low glucose concentration. Also, sfGFP with LVA is a better reporter for PcstA and PcsiE (Figure 7B).
After adding the LVA tag, the relative FI/OD600 decreased since the shorter half-life. Promoter
activity fold changes at different glucose concentrations were also increased with the addition of the LVA tag.
So we choose sfGFP with LVA as the reporter in experiments of Figure 8 and Figure 9. Unexpectedly, LVA tag
increases the relative FI/OD600 of PyciG abnormally (Figure 7A). This may be due to LVA tag
competitively inhibiting the degradation of sigmaS, for RpoS degrades in the same pathway with LVA-tagged
proteins.[2] Another interesting finding is on J23101. It is usually known as a constitutive
promoter, but its activity changes with the global metabolism level. At high glucose concentrations, the
activity of J23101 was significantly higher than that at low glucose concentration.

![](https://static.igem.wiki/teams/4115/wiki/results-pycig-with-lva.png)
![](https://static.igem.wiki/teams/4115/wiki/results-pcsta-pcsie-with-lva.png)

**Figure 7.** Relative FI/OD600 of the three selected starvation promoters. (A) PyciG. (B)
PcstA and PcsiE. J23101 (a constitutive promoter) is used as the positive control. Empty in Figure 2 is used
as the negative control and the automatic FI/OD600 of Empty can seem as a background signal. The
background signal has been removed from FI/OD600 of all groups shown in this figure. Then these
data are normalized with J23101 (under 4.0 g/L glucose) and shown as relative FI/OD600. (LVA)
indicates that in this group, the reporter is sfGFP with LVA. All the constructs were cloned into pUC
high-copy number backbone.

We mainly evaluate the quality of promoters from **two aspects**:
First, **promoter activity** is an
important evaluation index. Empirically, we believe that promoter activity not lower than the J23101 (a
constitutive promoter with moderately strong activity) is necessary for production and genetic circuits. Second,
**fold-change** is another important perspective for promoters, especially for those being used in
complicated genetic circuits. Fold-change can be defined as the ratio of the activity in the activated state to
the non-activated state. For our starvation promoters, the practical definition is the ratio of promoter
activity under low glucose concentration to that under high glucose concentration. We want the fold change to be
as large as possible.
According to the above data in Figure 7, we find that PcstA has the largest fold-change
and proper activity among the three starvation promoters. To further increase its fold change and activity, we
make some mutants on its CRP-binding sequence and Fis-binding sequence (Figure 8). To see the detailed design
strategy of these mutants, please go to our [Improve](https://2022.igem.wiki/shanghaitech-china/improve) page.

![](https://static.igem.wiki/teams/4115/wiki/results-pcsta-variants.png)

**Figure 8.** Regulation-related sequences of PcstA variants, the mutant bases are marked with bright blue.

We first cloned all of the report gene constructs into pUC high-copy number backbone to see if our mutants have
better properties. **E. coli** transformed with these constructs are cultured in M9 minimal medium
with different glucose concentrations. A significant promoter activity increase was observed on PcstA\_Mutant1
(Figure 9A). However, some unwanted phenomena also appeared. First, the leakage expression of PcstA\_Mutant1
under high glucose concentration increases, which results in the decrease of fold-change. Second, the high copy
of PcstA\_Mutant1 in cells has some side effects on growth (Figure 9B). We hypothesize that the high copy of
PcstA\_Mutant1 causes these side effects. CRP regulates the gene expression related to central metabolisms like
glycolysis and pentose phosphate pathway. For example, CRP activates the expression of *glk*(glucokinase),
which catalyzes the first step of glucose metabolism. So we suppose lack of CRP will decrease the glucose
utilizing of *E. coli*. The mutant on the CRP-binding sequence of PcstA\_Mutant1 increases its affinity to
CRP. If PcstA\_Mutant1 keeps a high copy in cells, it may compete the CRP with the endogenous CRP-dependent
promoter. Under the high glucose concentration, even though the abundance of CRP in cells is low, the high copy
and high affinity of PcstA\_Mutant1 cause the small amount of CRP to be still used to activate PcstA\_Mutant1.
This might be a possible explanation of the higher leakage activity of PcstA\_Mutan1. Less CRP proteins can be
used to regulate endogenous gene expression and cause some side effects on global metabolism[20].

![](https://static.igem.wiki/teams/4115/wiki/results-pcsta-on-puc.png)
![](https://static.igem.wiki/teams/4115/wiki/results-growth-decrease.png)

**Figure 9.** (A) FI/OD600 of PcstA variants. All the constructs are cloned into pUC high-copy
number backbone. The data analysis process is the same with Figure 7. (B) Growth curve of Empty and
PcstA\_Mutant1 strain in M9 minimal medium containing 4.0 g/L glucose as the sole carbon source.

Considering *E. coli* is the production chassis in our ternary microbial symbiosis system, side effects on
growth and global metabolism are serious problems. To eliminate the undesirable properties of PcstA\_Mutant1, we
shifted all constructions to the low-copy number pET backbone and repeat the experiment (Figure 10). Another
PcstA variant, PcstA\_Mutant2 is tested in this-time experiment. The bacteria growth is back to normal in all of
the groups, which may partially support our hypothesis on the growth. Mutants of PcstA show significantly higher
activities as well as larger fold-changes with low-copy number backbone. Compared with PcstA\_Mutant1,
PcstA\_Mutant2 shows higher activity and more obvious does-dependency. However, the leakage expression of
PcstA\_Mutant2 is higher than that of PcstA\_Mutant1, which results in a decrease in fold-change. The data of
relative promoter activities and fold changes of PcstA variants are summarized in Table 2.

![](https://static.igem.wiki/teams/4115/wiki/results-pcsta-mutants.png)

**Figure 10.** FI/OD600 of PcstA variants. All the constructs are cloned into pET low-copy
number backbone. The data analysis process is the same with Figure 7.

**In conclusion, after two cycles of optimization, by mutating the regulation-related sequence and replacing
the plasmid backbone, we finally get Pcst\_Mutant1 (on pET backbone) as the best construct for the nutrient
response.** We are never satisfied with the fold-change. For the future plan, we intend to use an
'amplifier' strategy to further optimize the properties of this nutrient response part.

![](https://static.igem.wiki/teams/4115/wiki/results-conclusion.png)

**Table 2.** Summarized data of PcstA variants. High [Glucose]=2 g/L, Low [Glucose]=0.25 g/L. Relative
promoter activities are normalized with FI/OD600 of J23101 at 2 g/L glucose.

Quorum sensing

We choose a widely used quorum sensing system, ***lux*** for this purpose. The whole *lux* system
can be separated into two parts: the signal sender and the signal receiver. 3O C6 HSL is the signal molecule of
quorum sensing. The signal sender expresses an AHL synthetase, LuxI ([BBa\_C0161](http://parts.igem.org/Part%3ABBa_C0161)). The signal receiver needs to express a composite
part device [BBa\_K4115039](http://parts.igem.org/Part%3ABBa_K4115039), which is mainly based on LuxR
([BBa\_C0062](http://parts.igem.org/Part%3ABBa_C0062)).
By design, the signal receivers should be
cyanobacteria and nitrogen fixation bacteria. However, we met some problems with expressing the receiver device
in those two chassis. To test the feasibility of our constructed quorum sensing system, we first expressed the
receiver device in *E. coli*.
To examine whether the signal receiver can function properly, we induced
the reporter (sfGFP) expression in the receiver with 3OC6 HSL at concentrations ranging from 10-4 to
10-14 M. After 3 hours induction, the cultures are used for measuring FI and OD600. The
data was fitted with a four-parameter logistical curve model as shown below.

`FI\text{/}OD\_{600}[3OC6 HSL]=b+\frac{a-b}{1+10^{((log(EC50)-log([3O C6 HSL]))\times h)}}`

a is the maximal sfGFP output, b is the basal sfGFP output, [3OC6 HSL] is the supplied inducer concentration,
sfGFP is the fluorescence response of the device for that inducer concentration, EC50 is the inducer
concentration that results in half maximal activation of the device, and h is proportional to the value of the
steepest slope along the curve (Hill coefficient) that indicates the responsiveness of the device to the
input.[7] Our data fitted the logistical curve successfully (Figure 11). Concentration-dependent
changes in promoter activity were observed between 10-10 and 10-7 M. 10-7 M
3OC6 HSL is sufficient to induce the maximum activity of lux pR ([BBa\_R0062](http://parts.igem.org/Part%3ABBa_R0062)).

![](https://static.igem.wiki/teams/4115/wiki/results-qs-receiver.png)

**Figure 11.** Induced sfGFP expression of the receiver with 3O C6 HSL at concentrations ranging from
10-4 to 10-14 M.

After confirming that our receiver is valid, we used the supernatant of the signal sender culture to activate
the signal receiver (Figure 12). 10-7 M 3O C6 HSL is supplied for positive control. The supernatant
successfully induced the receiver to produce efficient sfGFP expression compared to the negative control
(Blank). Referring to Figure 11, the concentration of 3O C6 HSL in the supernatant is about 10-9 M.

![](https://static.igem.wiki/teams/4115/wiki/results-qs-sender.png)

**Figure 12.** Induced sfGFP expression of the receiver with the supernatant of the signal sender culture.
Blank: Culture of receiver strain without treating with 3O C6 HSL

**In conclusion, these experiments demonstrate our quorum-sensing constructs can achieve intercellular
communication between bacteria.**

*S. elongatus*

Growth Curve

Since cyanobacteria are a brand new chassis for our team, we first explored how *S. elongatus* grow under
our culture conditions. We measure the growth curves of two strains, UTEX2973 (Figure 13) and HL7942 (Figure 14)
over 700 hours to test their growth situation.

![](https://static.igem.wiki/teams/4115/wiki/results-growthutex2973.jpg)

**Figure 13.** Growth curve of *S. elongatus* UTEX2973.

Since the strain used in the following experiments was *S. elongatus* HL7942, we focused on analyzing the
growth curve of *S. elongatus* HL7942. The curve reflects that its growth trend is closer to a straight
line rather than a logistic curve. This is mainly caused by cell-cell shading.[8] In other words,
because we put the flask with bacterial fluid into a horizontal shaking table for culture, there's poor mixing
of bacterial fluid at the centre and outer of the flask. The light received by the bacteria in the centre of the
flask is absorbed by the outer bacteria. Thus, with the centre of the flask as the centre of the circle, the
growth situation of *S. elongatus* is different at different radii. When we mixed the whole bacterial fluid
and measured the growth curve, its image was in a straight line. To see more details, please go to our [Model](https://2022.igem.wiki/shanghaitech-china/model) page.

Nutrient circulation

As an autotrophic microorganism, *S. elongatus* can produce biomass using CO2 as the substrate.
However, in natural conditions, *S. elongatus* prefer to store biomass for use but not provide for other
organisms. To overcome this problem, we integrated a sucrose permease, cscB expression box to *S.
elongatus* genome.

Sucrose production

**Construction of plasmid**

We expected that our *S. elongatus* can improve the ability to secrete sucrose by expressing CscB, sucrose
permease. In order to integrate CscB gene and the screening marker gene into the genome of *S. elongatus*,
we constructed a plasmid used to perform genetic manipulation on *S. elongatus* containing cscB gene and
kanamycin resistance gene located between the homologous arms upstream and downstream of the neutral site III.
We finally constructed a pUC57-NS3-2-Plac\_UV5-cscB-lacI-KanR-NS3-1 (Parts: [BBa\_K4115045](http://parts.igem.org/Part%3ABBa_K4115045)) plasmid (Figure 15).

![](https://static.igem.wiki/teams/4115/wiki/results-cscbplasmid.png)

**Figure 15.** Construction of pUC57-NS3-2-Plac\_UV5-cscB-lacI-KanR-NS3-1.

To complete this work, we conducted several molecular cloning experiments. We linearized the vector and
fragment to be used and recombined them by Gibson assembly.
The final constructed plasmid was sent for
sequencing and the results proved its correct sequence (Figure 16).

![](https://static.igem.wiki/teams/4115/wiki/results-plasmidcscbcheck.png)

**Figure 16.** The results of sequencing of the plasmid to be used for transformation.

**Transformation and recombination of *S. elongatus***

We used *S. elongatus* HL7942 for gene manipulation and coated two kanamycin-selective BG11 plates. After
5 days of culturing, several small colonies occurred. After 8 days of culturing, we picked two colonies of each
plate and inoculated them into the liquid medium (Figure 17).

![](https://static.igem.wiki/teams/4115/wiki/results-transformpalte.jpg)

**Figure 17.** Kanamycin-selective BG11 plates after 8 days of culturing. The area in the black circle is
the area for picking colonies.

After 18 days of culturing, we obtained *S. elongatus* that reached the logarithmic growth period. To test
whether the target gene, cscb and lacI, was successfully integrated into the *S. elongatus* genome, we did
colony PCR at different annealing temperatures to exclude non-specific binding. The result of the agarose gel
electrophoresis is shown in Figure 18. We recovered the target bands of engineering bacteria from tracks whose
annealing temperature is 59℃ and 60℃ and sent them for sequencing. The results showed that we successfully
inserted the target gene with the correct sequence (Figure 19).

![](https://static.igem.wiki/teams/4115/wiki/results-pcrcscb.png)

**Figure 18.** Colony PCR of recombinant *S. elongatus* at different annealing temperature. The area
shown in the red box is the target band area. As the annealing temperature rises, non-specific binding
reduces. The upper and lower figures are cut from the same gel map for easy annotation and observation.

![](https://static.igem.wiki/teams/4115/wiki/results-finalcheck.png)

**Figure 19.** The results of sequencing the recovered products. It shows that there's no mutation on CscB
or qlacI.

**Test on the capacity of engineering *S. elongatus* to produce sucrose**

In order to test the ability of the engineering bacteria, we set several control groups and experiment groups
under different conditions. These conditions included whether 1mM IPTG or 100mM NaCl existed in the medium. Set
three repetitions for each group. We detected the sucrose concentration of supernatant and OD685 value every 24
hours and lasted for 3 days. We compared the accumulation of sucrose secreted by a unit number of *S.
elongatus* in a certain period of time by comparing the ratio of sucrose concentration in the supernatant
to OD685 value of *S. elongatus* (Figure 20).

![](https://static.igem.wiki/teams/4115/wiki/results-induced24h.png)

**Figure 20A.** The ratio of sucrose concentration in the supernatant to OD685 value after
inducing 24 hours.

![](https://static.igem.wiki/teams/4115/wiki/results-induced48h.png)

**Figure 20B.** The ratio of sucrose concentration in the supernatant to OD685 value after
inducing 48 hours.

![](https://static.igem.wiki/teams/4115/wiki/results-induced72h.png)

**Figure 20C.** The ratio of sucrose concentration in the supernatant to OD685 value after
inducing 72 hours.

**Figure 20.** The ratio of sucrose concentration in the supernatant to OD685 value after
inducing.

The results show that under any conditions, engineering bacteria have a better capacity for sucrose production
than wild-type ones. In addition, engineering bacteria have the best capacity when 1mM IPTG and 100mM NaCl exist
at the same time among all of the different conditions. In order to understand the changing trend of the
capacity for sucrose production of engineering bacteria more intuitively over time, we made a graph based on the
ratio of the capacity of engineering bacteria and wild-type ones (Figure 21).

![](https://static.igem.wiki/teams/4115/wiki/results-inducedratio.png)

**Figure 21.** The ratio of the capacity of engineering bacteria and wild-type bacteria under different
conditions and different times they took for induction.

The figure shows that the ratios under each condition increase over time except for the one with both IPTG and
NaCl, yet the latter begins to increase after 48 hours of culturing. The reason for this phenomenon may be
caused by cscb. As a sucrose permease, cscB can not only transport sucrose from intracellular to extracellular
but also reverse transport. As a co-transporter protein, CscB co-transports protons with sucrose along the
proton concentration gradient. During the first 24 hours, engineering *S. elongatus* secreted so much
sucrose and H+ that may greatly change the liquid surrounding. As a result, during the second 24
hours, engineered *S. elongatus* may import some sucrose and grow much faster. Thus, the ratio went down.
However, this problem will not occur in our designed system for our culture system is a large mobile phase. The
surrounding of the liquid won't be changed that greatly.

Intercellular genetic circuit

Response of intercellular signals

To study the response of *S. elongatus* to signal molecules, we decided to integrate Plux and LuxR into
*S. elongatus* genome. LuxR gene encodes R-protein that can receive the signal of hunger, AHL, and Plux is
a promoter that is activated by LuxR in concert with HSL. In addition, we chose sfGFP as an reporter protein
used to indicate the intensity of Plux. In addition. In order to integrate LuxR gene, the screening marker gene,
and sfGFP gene with Plux into the genome of *S. elongatus*, we constructed a plasmid used to perform
genetic manipulation on *S. elongatus* containing genes or sequences above located between the homologous
arms upstream and downstream of the neutral site I. We finally constructed a
pUC57-NS3-2-KanR-J23101-B0034-LuxR-B0015-Lux pR-B0034-sfGFP-B0015-NS3-1 (Parts:
[BBa\_K4115046](http://parts.igem.org/Part%3ABBa_K4115046)) plasmid (Figure 22).

![](https://static.igem.wiki/teams/4115/wiki/results-lurplasmid.png)

**Figure 22.** Construction of pUC57-NS3-2-KanR-J23101-B0034-LuxR-B0015-Lux pR-B0034-sfGFP-B0015-NS3-1

We linearized vector pUC57-NS3-2-NS3-1 and fragment KanR-LuxR-sfGFP for Gibson assembly. The agarose gel
electrophoresis results of linearization are shown in Figure 23. Then the constructed plasmid is sent for
sequencing. The results showed that we successfully constructed this plasmid (Figure 24).

![](https://static.igem.wiki/teams/4115/wiki/results-luxr.png)

**Figure 23.** Linearized vector pUC57-NS3-2-NS3-1 (NS3-Vec in the figure) and fragment KanR-LuxR-sfGFP
(K-LuxR-sfGFP-Frag in the figure).

![](https://static.igem.wiki/teams/4115/wiki/results-luxrcheck.png)

**Figure 24.** The results of sequencing.

We transformed *S. elongatus*-cscB using this plasmid. As of the writing of this text, no colony has been
produced in the selected culture medium. Due to the slow growth of *S. elongatus* and time constraints, we
were not able to repeat the experiment again.

*A. caulinodans*

In the system, *A. caulinodans* is responsible for nitrogen fixation by nitrogenase. *A. caulinodans*
is different from many other rhizobia. It can perform nitrogen fixation in the free state while other rhizobia
only do this when are symbiotic with plants[9, 10]. Besides, *A. caulinodans* has been
relatively well studied and genetically modified in previous literature, which provides us with sufficient
experience and guidance to carry out the operation[11, 12]. Therefore, we chose *A. caulinodans*
ORS571 strain as our final nitrogen provider in the project. We got the *A. caulinodans* ORS571 (AmpR+)
strain from Professor Huawei Liu of College of Life Sciences, Northwest A & F University as a gift.

Electrotransformation of *A. caulinodans*

All subsequent experimental validation about *A. caulinodans* ORS571 should be based on successful genetic
operations of it. The genetic operation of microorganisms always involves the transformation of heterologous DNA
fragments into the cell. In most previous literature, the transformation operation for *A. caulinodans* has
been triparental mating, which is considered a complex and difficult method to operate. By contrast,
electrotransformation is a much simpler and less time-consuming method. We performed eletrotransformation to
transform the pBBR1MCS-2 (KanR) into *A. caulinodans* ORS571 cells as proof of successful genetic operation
(Figure 25). By reviewing the literature and summarizing the methods, we got a reliable protocol for the
electrotransformation of *A. caulinodans* and did experimental validation[13]. To see more
details about the electrotransformation, you can go to the [Experiments](https://2022.igem.wiki/shanghaitech-china/experiments) Page.

![](https://static.igem.wiki/teams/4115/wiki/reults-electro-colonies.jpg)

**Figure 25.** The single colonies of electrotransformed *A. caulinodans* on TY solid medium plate.
The pBBR1MCS-2 (KanR) plasmid was transformed into *A. caulinodans* cells. Two days later, single
colonies grew on the TY solid medium containing 30 μg/mL kanamycin.

Intercellular genetic circuit

In the nutrition regulation genetic circuit, *A. caulinodans* acts as the nitrogen output, which receives
intercellular signals to control its output rate. This requires two aspects: one is the response of
intercellular signals, and the other is the controllable regulation of nitrogen fixation rate.

Response of intercellular signals

We constructed the LuxR-lux pR-sfGFP sequence into pBBR1 backbone. The LuxR-lux pR-sfGFP sequence can respond
to the 3O C6 HSL signal, which has been demonstrated in *E. coli*. The broad-spectrum shuttle plasmid of
Gram-negative bacteria, pBBR1, can replicate in *A. caulinodans*. By our construction, we hoped to make
"pBBR1-luxR-lux pR-sfGFP" transformed *A. caulinodans* be able to respond to intercellular signal molecule
3O C6 HSL.

[![](https://static.igem.wiki/teams/4115/wiki/results-pbbr1-lux-sfgfp.png)](https://static.igem.wiki/teams/4115/wiki/results-pbbr1-lux-sfgfp.png)

**Figure 26.** The Snapgene map and file of the plasmid pBBR1-luxR-Plux-sfGFP. This plasmid was built for
response to intercellular signal molecule 3O C6 HSL in *A. caulinodans*. So after transforming the
plasmid into *A. caulinodans*, the green fluorescence intensity would show an upward curve with the
concentration of 3OC6HSL. The luxR system had been proven workable in some common chassis like *E. coli*,
so we did this experiment to figure out whether the luxR system would be workable as well in *A.
caulinodans*.

After PCR validation, pre-experiments of fluorescence measurement were performed. The results showed that the
luxR system seemed to be not workable in *A. caulinodans*.

![](https://static.igem.wiki/teams/4115/wiki/results-a-caul-lux.png)

**Figure 27.** The luxR response curve in *A. caulinodans*. Under the induction of a gradient
concentration of 3OC6HSL, there is no obvious fluorescence compared to the negative control(with no
induction).

We transformed the same plasmid into *E. coli* and performed trial induction, of which the result was good,
so there was no problem with the sequence itself.

![](https://static.igem.wiki/teams/4115/wiki/results-pbbrlux-e-coli.png)

**Figure 28.** The pre-experiment of trial 3OC6HSL induction and fluorescence measurement in
"pBBR1-luxR-Plux-sfGFP" transformed *E. coli*.

We assumed that the RNA polymerase in *A. caulinodans* is unable to interact with luxR protein, which makes
the luxR system fail to respond to signaling molecules. Thankfully, previous articles have already had
successful results on the intercellular signal response in *A. caulinodans* through other signal
systems[11, 12]. So our concepts in this part can also be proved by reference. In the future, we plan
to optimize our experimental conditions and change our signal system for our independent proof of concept.

Controllable regulation of nitrogen fixation Rate

![](https://static.igem.wiki/teams/4115/wiki/descriprtion-a-cregultae.png)

**Figure 29.** The schematic diagram of controllable regulation of nitrogen fixation rate.

In *A. caulinodans*, the nitrogen fixation is tightly controlled by the concentration of intracellular
ammonium and this counts for the nitrogenase master regulator *nifA*[14]. To achieve
controllable regulation of nitrogen fixation rate, endogenous regulators must be removed. In previous research,
knocking out of genomic *nifA* gene and then complementing by *nifA* controlled by inducible promoters
was proved to be a viable strategy[11, 12]. So we first transformed the suicide plasmid into *A.
caulinodans* to construct the *ΔnifA* strain, and then we transformed the shuttle plasmid into the
*ΔnifA* strain to complement the inducible *nifA* gene.
We learned from research that the knockout
of a target gene can be achieved by inserting a DNA sequence into a specific site on the genome using homologous
recombination. Therefore, we got the genome sequence of *A. caulinodans* ORS571 from Genbank and designed
the suicide plasmid ([BBa\_K4115041](http://parts.igem.org/Part%3ABBa_K4115041)).

![](https://static.igem.wiki/teams/4115/wiki/results-suicide-plasmid.png)

**Figure 30.** The Snapgene map and file of the plasmid pUC-UBRD . We synthesized the 900bp sequence
upstream and downstream of *nifA* on the genome as the homologous arm. Then, we took the bleomycin
resistance gene BleoR as the selection mark and constructed it on the plasmid between the two homologous arms.
All these fragments were cloned into the pUC plasmid. The final suicide plasmid was named pUC-UBRD
(Upstream\_homologous\_arm-BleoR-Downstream\_homologous\_arm).

The pBR322 origin of the suicide plasmid cannot drive a replication in *A. caulinodans*. The only way the
BleoR gene can be passed on as bacteria proliferates is to be integrated into the genome. Therefore, after
eletrotransforming the suicide plasmid into *A. caulinodans* and performing the bleomycin selection, the
colonies growing on the plate are likely to be the *ΔnifA* strain. To verify positive colonies, colony PCR
was performed.

![](https://static.igem.wiki/teams/4115/wiki/results-primers-knockout.png)

**Figure 31.** The primers of colony PCR to verify *ΔnifA* colonies. Two pairs of primers were chosen.
One pair of primers bind to the upstream and downstream homologous arm sequences to identify if BleoR was
inserted into the genome. The other pair of primers bind to the *nifA* coding sequence (CDS) to identify
if the *nifA* gene was knocked out.

After colony PCR, one *ΔnifA* colony was found. The gel map of the PCR result is shown in Figure 32. After
preliminary analysis, the BleoR sequence has been inserted into the genome, and the *nifA* gene has been
knocked out. But there are still some phenomena that cannot be explained well just by PCR and electrophoresis.
The Band-Ⅲ coming from the negative control template wild-type genome seems to have a similar length as Band-Ⅰ
and Band-Ⅳ but much shallower; Both the wild-type and *ΔnifA* genome lanes have smaller non-specific Band-Ⅳ
and Band-Ⅴ; The Band-Ⅶ coming form the template *ΔnifA* genome, seems to have the similar length as Band-Ⅷ,
but much shallower. So further sequencing validation to figure out what all of these bands are is necessary.

![](https://static.igem.wiki/teams/4115/wiki/results-gel-knockout.png)

**Figure 32.** Colony PCR of Δ*nifA* colony. For the PCR reaction whose primers bind on the homologous
arm sequences: Band-Ⅰ coming from the positive control template pUC-UBRD, is the BleoR sequence; Band-Ⅱ coming
from the negative control template wild-type genome, is the *nifA* sequence; Band-Ⅵ coming from the
Δ*nifA* genome template, is thought to be the inserted BleoR sequence because it has similar length as
Band-Ⅰ. For the PCR reaction whose primers bind on the *nifA* coding sequence: Band-Ⅷ coming from the
negative control template wild-type genome, is the *nifA* sequence while the lane of the Δ*nifA*
genome template has no such band.

Luckily, all the unwanted bands have been identified by DNA sequencing as other sites on the genome which is far
from *nifA*. The Band-Ⅳ has been identified as the BleoR gene. The results show that the gene knock-out has
been achieved, and the *ΔnifA* strain has been constructed.

![](https://static.igem.wiki/teams/4115/wiki/results-a-c-map.png)

**Figure 33.** The location of the bands of Colony PCR of Δ*nifA* colony after DNA sequencing. All the
unwanted bands have been identified by DNA sequencing as other sites on the genome which is far from
*nifA*.

Due to limited laboratory conditions, we have not been able to do a direct characterization of nitrogenase
activity. Through literature review, we found an alternative plan. The nifH gene expresses a subunit of
nitrogenase whose promoter PnifH is tightly controlled by transcriptional activators *nifA*. Therefore, the
part PnifH-sfGFP can effectively reflect the expression of nitrogenase and the content of *nifA*. We chose
the broad-spectrum shuttle plasmid of Gram-negative bacteria, pBBR1 as the backbone, so the plasmid can
replicate in *A. caulinodans*. Therefore, the shuttle plasmid pBBR1-PnifH-sfGFP was built for
characterization of the change of nitrogenase expression by knock-out of genomic *nifA* gene ([BBa\_K4115042](http://parts.igem.org/Part%3ABBa_K41150421)).

![](https://static.igem.wiki/teams/4115/wiki/results-nifhsfgfp.png)

**Figure 34.** The Snapgene map and file of the plasmid pBBR1-PnifH-sfgfp. This plasmid was built for the
characterization of the change of nitrogenase expression by knock-out of the genomic *nifA* gene. So
after transforming the plasmid into A. caulinodans wild-type strain and Δ*nifA* strain, the green
fluorescence intensity would show a significant decrease in Δ*nifA* strain, compared to the wild type.

After knocking out the genomic *nifA* gene, the regulation of nitrogen fixation in *A. caulinodans*
should be achieved by controllable *nifA* expression. In order to do that, we constructed the composite
part Plac-nifA-LacI to make *nifA* gene under IPTG induction. Moreover, the part PnifH-sfGFP was added to
reflect the expression of nitrogenase and the content of *nifA*. Therefore, the shuttle plasmid
pBBR1-PnifH-sfgfp-Plac-*nifA*-LacI ([BBa\_K4115043](http://parts.igem.org/Part%3ABBa_K4115043)) was
built for regulation of nitrogenase expression and characterize such expression .

![](https://static.igem.wiki/teams/4115/wiki/results-nifhsfgfp-lacnifa.png)

**Figure 35.** The Snapgene map and file of the plasmid pBBR1-PnifH-sfgfp-Plac-nifA-LacI. This plasmid was
built for regulation of nitrogenase expression and characterize such expression. So after transforming the
plasmid into *A. caulinodans* Δ*nifA* strain and giving IPTG induction, the green fluorescence
intensity would show an upward curve with the concentration of IPTG.

After PCR validation and pre-experiment of fluorescence measurement, there was no positive result. We repeated
a few times, but unfortunately, all the experiments failed.

At first, there were no positive bands for the experimental group in PCR validation. Some colonies did grow
under kanamycin selection, and a negative control plate with no colony showed that the kanamycin did work. So we
assumed that those plasmids were transformed into cells (since kanamycin selection worked), but systematic false
negatives occurred in colony PCR. Maybe the DNA polymerase worked badly in colony PCR of *A. caulinodans*.
Then, we directly carried out the pre-experiment of trial IPTG induction and fluorescence measurement of the
colonies. The results showed that the "pBBR1-PnifH-sfGFP" transformed cells (both wild-type and *ΔnifA*)
showed no obvious fluorescence, and the "pBBR1-PnifH-sfGFP-Plac-nifA-LacI" transformed *ΔnifA* cells showed
no obvious fluorescence after induction of adequate IPTG.

Due to the time limitation, the experiment had to end though there was no successful result. Thankfully,
previous articles have already had successful results in the same experiment, so our concepts in this part can
also be proved by reference[11, 12]. In the future, we plan to optimize our experimental conditions
for our independent proof of concept.

Immobilization and Co-Culture

The separate-immobilized fermentation pattern we proposed should be how the three microbial symbiotic system is
implemented in practice. To prove this concept, the three microorganisms should survive under some of the same
medium condition, immobilized cells should remain viable for a period of time, and material exchange between
immobilized cells needs to be verified. So we first set up a medium condition in which three microorganisms
could separately live. Then, microorganisms were immobilized and tested for viability in the gel. After that,
all three immobilized microorganisms were cultured in a container, under the same culture medium, and tested for
viability. Lastly, the material exchange between immobilized cells was validated.

Separate Culture in the Same Medium

Growth Curve of Separate Cultures in CoBG11-Sucrose Medium

We had known from previous research about the CoBG11 medium protocols for the coculture of Synechococcus and
other heterotrophic microorganisms. Based on this, we proposed our CoBG11-Sucrose medium protocols by adding
sucrose as solo carbon source to simulate the sucrose secretion of *S. elongatus*. Moreover, ammonium
chloride in the medium can be used as a simulation of ammonium production by *A. caulinodans*. To see the
ingredients of the culture medium, you can go to the [Experiments](https://2022.igem.wiki/shanghaitech-china/experiments) Page.

![](https://static.igem.wiki/teams/4115/wiki/results-separate1.png)

**Figure 36.** The growth curve of three microorganisms in CoBG11-Sucrose medium.

Through the resulting growth curve, we found that there is a significant rise in the growth curve of *E.
coli* and *S. elongatus* for a period of time (Figure 36.a&b). This indicated that *E. coli* and
*S. elongatus* can live and grow in CoBG11-Sucrose medium . However,the growth curve of *A.
caulinodans* quickly reached a plateau and remained at a low OD, which indicated that it cannot live and
grow in CoBG11-Sucrose medium (Figure 36.c). After research review, our hypothesis was confirmed. *A.
caulinodans* prefers to use organic acid as its carbon source rather than sugar[15]. Therefore,
when the medium contains only sugar as solo carbon source, *A. caulinodans* cannot live and grow.

Growth Curve of *A. caulinodans* in CoBG11-Acetate Medium

We found that *E. coli* would excrete significant amounts of acetate when growing aerobically on glucose as
the sole carbon source, which SacC-positive *E. coli* growing on sucrose will be in a similar
situation[16]. In this case, *A. caulinodans* can take acetate as its solo carbon source.
Therefore, we proposed a new CoBG11-Acetate protocol by adding acetate as solo carbon source to simulate the
acetate secretion of *E. coli*. In this experiment, we culture the *A. caulinodans* both in
CoBG11-Sucrose and CoBG11-Acetate to prove that the change of carbon sources can make *A. caulinodans*
successfully live in CoBG11 medium. To see the ingredients of the culture medium, you can go to the [Experiments](https://2022.igem.wiki/shanghaitech-china/experiments) Page.

![](https://static.igem.wiki/teams/4115/wiki/results-a-c-carbon.png)

**Figure 37.** The growth curve of *A. caulinodans* in CoBG11-Sucrose/Acetate medium.

After replacing the carbon source, the growth of *A. caulinodans* was significantly improved. The result
confirms that *A. caulinodans* can live and grow in CoBG11 medium when acetate is the solo carbon source.

Immobilization of Microorganisms

To verify the feasibility of "Separate-Immobilized Fermentation", a preliminary immobilization method needs to
be designed. After research, we got an achievable protocol, which immobilizes the cells by calcium alginate
embedding[17]. Then, immobilized cells were periodically sampled and lysed through phosphate buffer,
and the OD value of lysate was measured to reflect the viability of immobilized cells (Figure 39.d). Besides,
for bacteria expressing the green fluorescent protein, fluorescence intensity can be obtained by taking
fluorescence images of the lysate to reflect viability. To see more details about the immobilization, you can go
to the [Experiments](https://2022.igem.wiki/shanghaitech-china/experiments) Page.

Immobilization of *E. coli* and *S. elongatus*

![](https://static.igem.wiki/teams/4115/wiki/results-drip-device.jpg)

**Figure 38.** Simple drip device. It helps to fix the syringe and make alginate-cell mixture drips
naturally into calcium chloride solution.

![](https://static.igem.wiki/teams/4115/wiki/results-immo-e-s.png)

**Figure 39.** Immobilized *E. coli* and *S. elongatus*. (a)&(b) Immobilized *E. coli*
(pUC-J23101-sfGFP). (a) is under blue light, the immobilized *E. coli* emits green fluorescence; (b) is
under natural light, the immobilized *E. coli* appears white. (c) Immobilized *S. elongatus*. It is
under natural light, the immobilized *E. coli* appears green. (d) The lysate of the immobilized *E.
coli* and *S. elongatus*.

Survival Curve of the Immobilized *E. coli* and *S. elongatus*

After a week of lysis of immobilized cells and OD or fluorescence measurement, both OD survival and fluorescence
survival curves did not show a significant downward trend (Figure 40.a-c). The results showed that we had
successfully produced immobilized *E. coli* and *S. elongatus*, which maintained high viability in the
gel.

![](https://static.igem.wiki/teams/4115/wiki/results-immo-survive.png)

**Figure 40.** The OD survival curve of Immobilized *E. coli* and *S. elongatus*. (a) The
OD685 survival curve of immobilized *S. elongatus*. (b)&(c) The OD600 and
fluorescence survival curve of immobilized *E. coli*. (d) The image of lysed immobilized *E. coli*,
was analyzed to get the fluorescence intensity.

Confocal Imaging of Immobilized *E. coli*

Besides, the sfGFP and tagRFP marked immobilized *E. coli* were observed under a fluorescent confocal
microscope. The result would help us to have a clear idea what a single cell is like when being immobilized in
calcium alginate gel.

![](https://static.igem.wiki/teams/4115/wiki/results-confocal.png)

**Figure 41.** The Confocal images of immobilized *E. coli*. The immobilized *E. coli* in calcium
alginate gel was colocalized by fluorescence. They are fairly evenly distributed in the gel beads, and the
contour is clear.

Co-culture of Immobilized Microorganisms

Survival Curve of Co-cultured Immobilized Microorganisms

To verify the feasibility of the "Separate-Immobilized Fermentation", three microorganisms need to be
immobilized and cultured in the same medium condition. In past experiments, it has been demonstrated that *E.
coli* can survive in CoBG11 medium containing sucrose as a solo carbon source, while *A. caulinodans*
can survive in CoBG11 medium containing acetate as a solo carbon source. In this experiment, sucrose and acetate
were added to the CoBG11 medium (called CoBG11-PLUS) to simulate the sucrose secretion of *S. elongatus*
and the acetate secretion of *E. coli*. To see the ingredients of the culture medium, you can go to the [Experiments](https://2022.igem.wiki/shanghaitech-china/experiments) Page.

The immobilized *E. coli* expressing red fluorescent protein appears faint red under natural light,
immobilized *A. caulinodans* appears white, and immobilized *S. elongatus* appears green. By color, we
can distinguish three immobilized microorganisms, and then sample and measure the OD value to draw the inventory
curve.

![](https://static.igem.wiki/teams/4115/wiki/results-three-immo.jpg)

**Figure 42.** Immobilized *E. coli*, *A. caulinodans* and *S. elongatus* in the same
CoBG11-PLUS medium. tagRFP-marked *E. coli* appears faint red, *A. caulinodans* appears white and
*S. elongatus* appears green.

After a week of lysis of immobilized cells and OD measurement, OD survival curves did not show a significant
downward trend during a week. The results showed that all three immobilized microorganisms can maintain high
viability in the gel and in the same medium condition.

![](https://static.igem.wiki/teams/4115/wiki/results-three-immo-survive.png)

**Figure 43.** The survival curve of three immobilized microorganisms.

Substance Circulation Between Immobilized Cells

The effective substance exchange between immobilized cells is important because all the symbiotic nutrition
relationships and intercellular genetic circuit regulation rely on this exchange. We did experimental validation
of the intercellular communication in immobilized *E. coli* through the LuxR system as a demo of
intercellular signal communication of our system as well as an intuitive proof of concept that substances can
circulate between immobilized cells.

Intercellular Communication in Immobilized *E. coli* Through LuxR System

In this experiment, we applied Quorum sensing LuxR system to the intercellular communication in immobilized
*E. coli*. There were two kinds of immobilized *E. coli*: one carries the LuxI gene, which can
synthesize the signal molecule 3OC6HSL; the other kind of *E. coli* carries the LuxR-Plux-sfgfp sequence,
which can respond to the 3OC6HSL signal by expressing sfGFP and emit green fluorescence under blue light. The
above two kinds of immobilized *E. coli* were placed in the same medium to verify the intercellular
communication in immobilized microorganisms. Meanwhile, *E. coli* carrying no LuxI gene and *E. coli*
carrying the LuxR-Plux-sfGFP sequence were placed together to be the negative control.

After two days of co-culture and induction, the two experimental groups showed obvious fluorescence enhancement
compared with the negative control groups, which indicated the success of intercellular communication in
immobilized *E. coli*.

![](https://static.igem.wiki/teams/4115/wiki/results-lux-immo.png)

**Figure 44.** Intercellular communication in immobilized *E. coli* through LuxR system. The top
dishes are the negative control group while the bottom dishes are the experimental groups. There are two
parallel groups.

References

[1] Lee, J.W., Choi, S., Park, J.H., Vickers, C.E., Nielsen, L.K., and Lee, S.Y. (2010). Development of
sucrose-utilizing Escherichia coli K-12 strain by cloning beta-fructofuranosidases and its application for
L-threonine production. Appl Microbiol Biotechnol 88, 905-913. 10.1007/s00253-010-2825-7.

[2] Battesti, A., Majdalani, N., and Gottesman, S. (2011). The RpoS-mediated general stress response in
Escherichia coli. Annu Rev Microbiol 65, 189-213. 10.1146/annurev-micro-090110-102946.

[3] Franchini, A.G., Ihssen, J., and Egli, T. (2015). Effect of Global Regulators RpoS and Cyclic-AMP/CRP on
the Catabolome and Transcriptome of Escherichia coli K12 during Carbon- and Energy-Limited Growth. PLoS One 10,
e0133793. 10.1371/journal.pone.0133793.

[4] Rozen, Y., Dyk, T.K., LaRossa, R.A., and Belkin, S. (2001). Seawater Activation of Escherichia coli Gene
Promoter Elements: Dominance of rpoS Control. Microb Ecol 42, 635-643. 10.1007/s00248-001-1012-x.

[5] Gasperotti, A., Going, S., Fajardo-Ruiz, E., Forne, I., and Jung, K. (2020). Function and Regulation of the
Pyruvate Transporter CstA in Escherichia coli. Int J Mol Sci 21. 10.3390/ijms21239068.

[6] Marschall, C., and Hengge-Aronis, R. (1995). Regulatory characteristics and promoter analysis of csiE, a
stationary phase-inducible gene under the control of sigma S and the cAMP-CRP complex in Escherichia coli. Mol
Microbiol 18, 175-184. 10.1111/j.1365-2958.1995.mmi\_18010175.x.

[7] Kylilis, N., Tuza, Z.A., Stan, G.B., and Polizzi, K.M. (2018). Tools for engineering coordinated system
behaviour in synthetic microbial consortia. Nat Commun 9, 2677. 10.1038/s41467-018-05046-2.

[8] Broddrick, J.T., Rubin, B.E., Welkie, D.G., Du, N., Mih, N., Diamond, S., Lee, J.J., Golden, S.S., and
Palsson, B.O. (2016). Unique attributes of cyanobacterial metabolism revealed by improved genome-scale metabolic
modeling and essential gene analysis. Proc Natl Acad Sci U S A *113*, E8344-E8353.

[9] Jiang, N., Liu, W., Li, Y., Wu, H., Zhang, Z., Alexandre, G., Elmerich, C., and Xie, Z. (2016). A
Chemotaxis Receptor Modulates Nodulation during the Azorhizobium caulinodans-Sesbania rostrata Symbiosis. Appl
Environ Microbiol 82, 3174-3184.

[10] Lee, K.-B., De Backer, P., Aono, T., Liu, C.-T., Suzuki, S., Suzuki, T., Kaneko, T., Yamada, M., Tabata,
S., Kupfer, D.M., et al. (2008). The genome of the versatile nitrogen fixer Azorhizobium caulinodans ORS571. BMC
Genomics 9, 271.

[11] Ryu, M.H., Zhang, J., Toth, T., Khokhani, D., Geddes, B.A., Mus, F., Garcia-Costas, A., Peters, J.W.,
Poole, P.S., Ané, J.M., et al. (2020). Control of nitrogen fixation in bacteria that associate with cereals. Nat
Microbiol 5, 314-330.

[12] Haskett, T.L., Paramasivan, P., Mendes, M.D., Green, P., Geddes, B.A., Knights, H.E., Jorrin, B., Ryu,
M.-H., Brett, P., Voigt, C.A., et al. (2022). Engineered plant control of associative nitrogen fixation.
Proceedings of the National Academy of Sciences 119, e2117465119.

[13] 张俊彪 (2017). Azorhizobium caulinodans ORS571 Ⅲ型分泌系统效应蛋白的预测、鉴定及相关功能分析 (西北农林科技大学).

[14] Demtröder, L., Pfänder, Y., Schäkermann, S., Bandow, J.E., and Masepohl, B. (2019). *nifA* is the
master regulator of both nitrogenase systems in Rhodobacter capsulatus. Microbiologyopen 8, e921.

[15] 郝保海 (2012). 根瘤菌比较基因组学研究及蛋白质互作网络预测 (华中农业大学).

[16] Bernal, V., Castaño-Cerezo, S., and Cánovas, M. (2016). Acetate metabolism regulation in Escherichia coli:
carbon overflow, pathogenicity, and beyond. Applied Microbiology and Biotechnology 100, 8985-9001.

[17] 赵景联 (1989). 固定化大肠杆菌细胞生产γ-氨基丁酸的研究. 生物工程学报 5.

[18] Basan, M., Hui, S., Okano, H., Zhang, Z., Shen, Y., Williamson, J.R., and Hwa, T. (2015). Overflow
metabolism in Escherichia coli results from efficient proteome allocation. Nature 528, 99-104.
10.1038/nature15765.

[19] Pinhal, S., Ropers, D., Geiselmann, J., and de Jong, H. (2019). Acetate Metabolism and the Inhibition of
Bacterial Growth by Acetate. J Bacteriol 201. 10.1128/JB.00147-19.

[20] Shimada, T., Fujita, N., Yamamoto, K., and Ishihama, A. (2011). Novel roles of cAMP receptor protein (CRP)
in regulation of transport and metabolism of carbon sources. PLoS One 6, e20081. 10.1371/journal.pone.0020081.

{% endblock %}