iGEM 2024 — Team cau-china

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[notebook1](#1)

[notebook2](#2)

[notebook3](#3)

# notebook1

# notebook2

# notebook3

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![](https://static.igem.wiki/teams/5300/source/engineering/top.png)

[Wet lab engineering](#1)

[Dry lab engineering](#2)

# Wet lab engineering

## Regulation and synthesis module

The engineering cycle is divided into four phases: design, build, test, and learn. We design experiments according to our experimental purpose, build the system we designed using biological or other experimental methods, test the buildings using reasonable approaches, and finally learn as well as analyze the results of the testing and generate new questions and purposes so that we can move on to the next iteration.

We followed this principle during our experiments, and the engineering cycle helps us a lot to overcome difficulties. This page describes the five iterations we performed during the construction of the plasmids of the Synthesis circuit and the validation of the regulation module.

### 1st Iteration

#### DESIGN

The Synthesis module is divided into regulation module and *pfa* biosynthetic gene clusters. We planed to use the genome and plasmid as templates to amplify four elements of the regulation module using PCR. At the same time, we added homologous sequences to the 5' end of the primers, and finally assembled the four elements into the regulation module by overlap PCR.

On the other hand, we divided the extensive *pfa*biosynthetic gene cluster into five fragments (PUFA1-5) to be synthesized by biotech company. We incorporated the RBS sequences of *NifD*, *NifK*, *NifE* and *NifN* from *Sinorhizobium fredii* CCBAU45436 in front of the start codons to ensure compatibility with the *nifH* promoter. Furthermore, we optimized the original sequences according to the codon preference of *Sinorhizobium fredii* CCBAU45436 to enhance gene expression.

We also intended to initially fuse the regulation module with PUFA1, PUFA2 with PUFA3, and PUFA4 with PUFA5 using overlap PCR, followed by Gibson assembly to clone the fragments into pBBR1MCS-2 plasmid.

#### BUILD

We amplified *glnK* promoter and **nifH** promoter from the genome of *Sinorhizobium fredii* CCBAU45436 using PCR, and obtained *gfp* and tracrRNA from corresponding plasmid templates. We attached the sgRNA targeting the *gfp* sequence to the 5' end of the tracrRNA during PCR. By performing overlap PCR, we connected *glnK* promoter with sgRNA-GFP, then *nifH* promoter with gfp, and subsequently linked the two products through overlap PCR to yield the regulation module *glnK*-sgRNA-GFP-*nifH*-gfp. We used the plasmids of *pfa* biosynthetic gene clusters synthesized by the biotech company as templates to amplify the PUFA1-5 fragments through PCR, incorporating homologous sequences of adjacent fragments. We expected to achieve pairwise connections between the regulation module and PUFA1, PUFA2 and PUFA3, PUFA4 and PUFA5 through overlap PCR.

#### TEST

Our overlap PCR successfully yielded the regulation module *glnK*-sgRNA-GFP-*nifH*-*gfp* (Figure 1), with Sanger sequencing confirming the correct product.

Subsequently, we conducted overlap PCR experiments on three groups simultaneously, finding that none produced the desired fusion product bands. We employed Touchdown PCR to adjust the annealing temperature and attempted to modify extension time and primers, but the fusion PCR bands consistently exhibited severe trailing and strong non-specific amplification.

![](http://static.igem.wiki/teams/5300/source/engineering/1.png)

Figure1 Agarose gel electrophoresis for detection and purification of the regulatory sequence DNA fragment amplified by overlap PCR. The size of the regulatory sequence *glnK*-sgRNA-GFP-*nifH*-*gfp* should be 2211 bp, and we purified the DNA indicated in the white box using a gel recovery kit.

#### LEARN

We suspected that it may be due to the homologous arms being too short compared to the genes’ coding sequences for effective complementary linkage during annealing. Additionally, our PCR kit might not be suitable for complex templates amplification, which could explain the low efficiency of overlap PCR.

### 2nd Iteration

#### DESIGN

We abandoned the overlap PCR and opted for seamless cloning to ligate two fragments into the plasmid vector, and use the recombinant plasmids as templates for PCR amplification to obtain pairwise connected fragments. To achieve this, we designed three pairs of primers with corresponding fragment junctions on the MCS of the pUC19 plasmid. We linearized pUC19 via PCR, with the hope that the regulation module would first be joined with fragments of *pfa* biosynthetic gene cluster pairwise by seamless cloning.

Subsequently, we planned to use the recombinant plasmid as a template for PCR amplification to obtain regulation module-PUFA1, PUFA2-PUFA3, PUFA4-PUFA5, and then conduct seamless cloning with the linearized pBBR1MCS-2 plasmid, which was double digested with EcoRⅠ and XmaⅠ, to construct the final plasmid.

#### BUILD

We used Gibson assembly to connect the six fragments pairwise to the pUC19 plasmid vector and transformed *E. coli* (DH5α). After adding X-gal and IPTG to the culture, we plated it on LB solid medium containing ampicillin and incubated at 37°C overnight. We picked white colonies for overnight shaking in LB liquid medium with ampicillin. Plasmid extraction followed by EcoRⅠ single enzyme digestion and agarose gel electrophoresis (Figure 2) allowed us to select the correct plasmids: pUC19-regulation module-PUFA1, pUC19-PUFA2-PUFA3, as well as pUC19-PUFA4-PUFA5.

![](http://static.igem.wiki/teams/5300/source/engineering/2.png)

Figure2 Agarose gel electrophoresis for detecting correct recombinant plasmids. The extracted plasmids were digested by EcoRⅠ (TaKaRa) at 37°C for 10 minutes. The correct sizes of pUC19-regulation module-PUFA1 should be 8512 bp, at the same time, pUC19-PUFA2-PUFA3 and pUC19-PUFA4-PUFA5 should be 9799 bp and 9145 bp respectively. The bands indicated by boxes were of the correct sizes.

We selected plasmids 2, 5, and 8 for PCR amplification of the fused fragments. Meanwhile, we linearized the pBBR1MCS-2 plasmid using EcoR Ⅰ and Xma Ⅰ. Next, we used Gibson assembly to connect the linearized vector with the three fused DNA fragments.

#### TEST

Our PCR amplification successfully yielded correct sized fusion products (Figure 3), and we also obtained correctly sized bands from double digestion (Figure 4). We transformed the Gibson assembly products of the four fragments into *E. coli* (DH5α), plated them on LB solid medium containing kanamycin after adding X-gal and IPTG, and incubated at 37°C overnight. Both replicates yielded only one blue colony(Figure 5).

![](http://static.igem.wiki/teams/5300/source/engineering/3.png)

Figure3 Agarose gel electrophoresis for detecting and purifying the fusion DNA fragments obtained from PCR. Using plasmid 2 as template, we amplified the regulatory sequence-PUFA1 (5836 bp). Using plasmid 5, we amplified PUFA2-PUFA3 (7118 bp). Using plasmid 8, we amplified PUFA4-PUFA5 (6482 bp).

![](http://static.igem.wiki/teams/5300/source/engineering/4.png)

Figure4 Agarose gel electrophoresis for detecting and purifying the linearized pBBR1MCS-2 fragment obtained from double digestion. The pBBR1MCS-2 plasmid was digested with EcoR Ⅰ (NEB) and Xma Ⅰ (NEB) at 37°C for 15 minutes.

![](http://static.igem.wiki/teams/5300/source/engineering/5.png)

Figure5 LB-Agar plates. only one blue colony grew on both medium.

#### LEARN

We initially speculated that the low efficiency of multi-fragment Gibson assembly or the low transformation efficiency of large plasmid might be the cause. We adjusted the conditions for seamless cloning as well as temperature and time during transformation, but the results did not change; we also ruled out the possibility of problems with primer design.

Meanwhile, the unusual results of another plasmid constructed by the experimental group drew our attention. This recombinant plasmid inserted the *glnK*-*gfp* fusion product into the pBBR1MCS-2 plasmid vector to detect *glnK* promoter’s response to nitrogen. This plasmid vector was also linearized using EcoR Ⅰ and Xma Ⅰ, and the results from transformants colony PCR indicated that most of them contain empty plasmid. Given the high efficiency of the kit we used for single-fragment seamless cloning, such a situation was quite unusual.

Therefore, we speculated that there might have been low efficiency or even inactivation of the single enzyme during double digestion, resulting in extensive self-ligation of the vector and minor seamless cloning products. We suspected this could also explain the failure of the second iteration.

### 3rd Iteration

#### DESIGN

To investigate whether the restriction enzymes used for double digestion were functioning properly, we designed a validation experiment using single enzyme digestion. If a linearized plasmid band produced by a specific enzyme digestion exhibited low brightness while the corresponding band for the supercoiled plasmid showed high brightness, this would support our hypothesis.

#### BUILD

To verify the activity of the restriction enzymes, We employed the pBBRMCS-2 plasmid as a substrate and performed single enzyme digestions with EcoR Ⅰ and Xma Ⅰ under the same conditions as the second iteration.

#### TEST

The expected size for the linearized vector was 5132 bp, while the supercoiled plasmid typically exhibited a band around 3500 bp. The EcoR Ⅰ single enzyme digestion product showed a bright band at approximately 5000 bp with no band around 3500bp. Conversely, the Xma Ⅰ single enzyme digestion product displayed only a faint band at the same position, and the band of supercoiled plasmid is bright.

![](http://static.igem.wiki/teams/5300/source/engineering/6.png)

Figure6 Agarose gel electrophoresis used to detect band brightness from the pBBR1MCS-2 plasmid after single enzyme digestions with EcoR Ⅰ or Xma Ⅰ. The pBBR1MCS-2 plasmid (5132 bp) was digested with EcoR Ⅰ (NEB) or Xma Ⅰ (NEB) at 37°C for 15 minutes.

#### LEARN

The results of agarose gel electrophoresis indicated that the EcoR Ⅰ restriction enzyme exhibited normal activity. However, the activity of the Xma Ⅰ restriction enzyme was significantly low. This discrepancy resulted in very few recombinant products from our seamless cloning attempts. We speculated that repeated freeze-thaw cycles may have diminished enzyme activity. Given that the two restriction sites in the MCS were in close proximity, it was challenging to distinguish between single and double digestion products based on electrophoresis results, leading to a misinterpretation of the success of the enzyme digestion in the second iteration.

### 4th Iteration

#### DESIGN

Consequently, we abandoned the use of double digestion tor linearize the plasmid and designed the primers to linearize pBBR1MCS-2 using PCR. The linearized pBBR1MCS-2 was purified through gel extraction, and we employed Gibson assembly to combine the linearized pBBR1MCS-2 with the three fused DNA fragments, which were purified from the second iteration to construct the PUFA synthesis circuit plasmid.

#### BUILD

We obtained the correct bands through PCR and purified the linearized vector fragment using a gel extraction kit. Following the same protocol, we performed Gibson assembly and transformed the product into *E. coli* (DH5α). Then we selected white colonies for plasmid extraction.

![](http://static.igem.wiki/teams/5300/source/engineering/7.png)

Figure7 Agarose gel electrophoresis used to detect and purify the PCR-linearized pBBR1MCS-2 plasmid. Agarose gel (0.8%) was run at 150V for 15 minutes in 1x TAE buffer, stained with GelRed DNA gel stain, and Gel analysis was performed using blue light illuminator. The correct size for the linearized vector was expected to be 5148 bp, and we purified the band indicated by the box.

#### TEST

We successfully screened a correct clone on LB solid medium containing kanamycin , and the extracted plasmid was notably large, with an electrophoretic mobility slower than the 10000 bp band of 1 kb marker. Sanger sequencing results revealed a nonsense mutation in the open reading frame of *pfa*1, resulting in resulted in the termination codon appearing 113 amino acids earlier. Additionally, a frameshift mutation was detected in the *pfa*2 gene. Further sequencing of the corresponding mutation sites in the pUC19-regulation module-PUFA1 and pUC19-PUFA2-PUFA3 revealed that the former one was correct while the latter exhibited the same mutation.

![](http://static.igem.wiki/teams/5300/source/engineering/8.png)

Figure8 Agarose gel electrophoresis used to detect the recombinant pBBR1MCS-2 plasmid. Agarose gel (0.7%) was run at 150V for 15 minutes in 1x TAE buffer, stained with Safegreen DNA gel stain, and Gel analysis was performed using blue light illuminator. The size of the PUFA synthesis circuit recombinant plasmid is 24479 bp.

![](http://static.igem.wiki/teams/5300/source/engineering/9.png)

Figure9 Sequencing results. The purified PUFA synthesis circuit recombinant plasmid was subjected to commercial Sanger sequencing, with the original sequence indicated in bold. The bottom row represents the sequencing data, with red boxes denoting mutations.

#### LEARN

The mutation in *pfa*1 led to a partial deletion of the substrate-binding domain of the enoyl-CoA reductase (ER), while the mutation in *pfa*2 disrupted the expression of hydroxyacyl-CoA dehydrase (DH). These critical mutations necessitated the abandonment of this plasmid for validation experiments.

### 5th Iteration

#### DESIGN

Due to delays in gene synthesis, sequencing, and plasmid reconstruction, we lacked sufficient time to introduce the target plasmid into *Sinorhizobium fredii* CCBAU45436 who carried Cas12k gene and verify it on JiDou 17. Thus, we decided to separate the regulation module and *pfa* biosynthetic gene cluster to conduct vitro validation. For the regulation module, we planned to construct *PnuoA-Cas12k-Terminator*-regulation module into pBBR1MCS-2 plasmid. We intended to use triparental mating to introduce the plasmid into *Sinorhizobium fredii* CCBAU45436, and cultivate the resulting strains under high nitrogen-high oxygen, high nitrogen-low oxygen, low nitrogen-high oxygen, and low nitrogen-low oxygen conditions. By utilizing *nifH* promoter to regulate *gfp* expression, we would measure the fluorescence intensity and the OD600 ratio to quantitatively assess *gfp* expression, thereby validating the effectiveness of the regulation module.

As for the PUFA synthesis module, we aimed to construct *PnuoA*-*pfa* biosynthetic gene cluster into the pBBR1MCS-2 plasmid and introduce the plasmid into both *Sinorhizobium fredii*CCBAU45436. These strains would be cultured in nutrient-rich TY liquid medium, which would be used to conduct RT-qPCR to quantify expression of related genes.

#### BUILD

We reconstructed the aforementioned plasmids using existing parts, and confirmed their accuracy by Sanger sequencing. Employing triparental mating, we introduced the two recombinant plasmids into *Sinorhizobium fredii* CCBAU45436 respectively, spreading the mixed bacterial culture onto TY solid medium and incubating at 28°C for 48 hours. The resulting mixed bacterial sludge was diluted to and plated onto TY-TP-Kan solid medium, incubating at 28°C for 4 days. We pick single colonies and streaked them onto TY-TP-Kan solid medium. Using colony PCR, we selected three clones containing the recombinant plasmid, which involved *PnuoA-Cas12k-Terminator* and regulation module.

To prepare media with nitrogen concentration gradients, we substituted the TY medium with M9 medium supplemented with \(NH\_{4}Cl\) as the nitrogen source, allowing for quantitative adjustment of nitrogen levels. Based on the result of preliminary nitrogen respond experiment on *glnK* promoter, we formulated a low-nitrogen medium with 0 g/L \(NH\_{4}Cl\) and a high-nitrogen medium with 0.02 g/L \(NH\_{4}Cl\). Additionally, to create hypoxic conditions in vitro, we boiled the M9 medium, added melted petroleum jelly, and then cooled it on ice before injecting the bacterial culture into the medium. This mixture was incubating under shaking conditions at 28°C for 3 days. We designed four experimental groups based on oxygen and nitrogen levels, with three replicates for each group.

As for the PUFA synthesis module, we identified one clone containing the recombinant plasmid. After 2 days shaking incubation, we use RNA extraction and reverse transcription kit to obtained the cDNA from the +*pfa* strain and the wild-type strain. Meanwhile, we send bacteria solution for gas chromatographic identification.

#### TEST

##### Regulation module

We measured the relative fluorescence intensity of each group of bacterial fluids under excitation light at 488 nm using a fluorescence spectrophotometer. We also measured the OD600 value of each group of bacterial fluids using a spectrophotometer, as well as used the relative fluorescence intensity divided by the amount of expression of *gfp* representing individual bacterium, and the results were as shown in (Figure 10). Regardless of nitrogen concentration, the *gfp*nifH promoter can enhance gene expression in response to low oxygen concentration. Regardless of oxygen concentration, *gfp* expression at high nitrogen was significantly higher than that at low nitrogen (c,d). This suggests that our designed deterrent system can indeed increase sgRNA expression in response to elevated nitrogen, thereby suppressing downstream gene’s expression. Overall, the *gfp* expression at high nitrogen and low oxygen was significantly higher than the three other conditions, which is in line with expectations (Figure 11).

![](http://static.igem.wiki/teams/5300/source/engineering/10.jpg)

Figure10 Ratio of relative fluorescence intensity to OD600 value of bacterial. oxygen conditions at low nitrogen (0 g/L \(NH\_{4}Cl\)). (b) Different oxygen conditions at high nitrogen (0.02 g/L \(NH\_{4}Cl\)). (c) Different conditions of nitrogen concentration during hypoxia. (d) Different conditions of nitrogen concentration during hyperoxia. Student’s t-test, ns: no significant difference; \*, p-value < 0.05; \*\*, p-value&lt0.01 \*\*\*, p-value < 0.001.

![](http://static.igem.wiki/teams/5300/source/engineering/11.jpg)

Figure11 Overall ratio of relative fluorescence intensity to OD600 value of bacterial. Student’s t-test, ns: no significant difference; \*, p-value < 0.05; \*\*, p-value&lt0.01 \*\*\*, p-value < 0.001.

##### Synthetic module

As for PUFA synthesis module, using endogenous *nuoA* gene as contrast and 16SrRNA as internal reference, we detected the transcription level of *pfa* genes by RT-qPCR. The result shows that the transcription level of both *pfa* genes were significantly higher than *nuoA*, and the closer the gene near to the promoter, the higher the transcription level it detected (Figure 12).

As gas chromatographic result, most fatty acid level shows no significant difference between WT and +*pfa*, but there is a new fatty acid C14:0 been detected, which is never appeared in the wild type(Figure13).

![](http://static.igem.wiki/teams/5300/source/engineering/12.jpg)

Figure12 The relative transcription level between *nuoA* and *pfa* genes measured by RT-qPCR. Student’s t-test, ns: no significant difference; \*, p-value < 0.05; \*\*, p-value&lt0.01 \*\*\*, p-value < 0.001.

![](http://static.igem.wiki/teams/5300/source/engineering/13.jpg)

Figure13 The relative content of fatty acids measured by gas chromatography.

#### LEARN

##### Regulation module

Although the results were to some extent as expected as the model, we still detected a certain amount of *gfp* expression in the remaining conditions except for high nitrogen and low oxygen. The difference of the *gfp* between high nitrogen and low oxygen conditions with others was not as large as desired.

This might due to the fact that our Cas12k is not efficient enough for transcriptional repression, and there is a certain leakage expression from *glnK* promoter. Although our initial idea was making Cas12k to act as a terminator, according to the experimental results, it is still necessary to introduce a terminator after sgRNA, which will restrict the leakage expression of *glnK* promoter.

In addition, the design of sgRNA can likewise be improved. We can insert a variety of different sgRNA after *glnK* promoter, targeting different sequences of downstream genes. Whether it can also improve the efficiency of the Cas12k deterrent system is also a worthwhile area to be verified and improved.

Further more, lab condition may not intimate the natural environment of nodule sufficiently. For example, legume produce soybean hemoglobin for rhizobium, which can chelated oxygen to maintain a microaerobic environment for nitrogen fixation. Whereas our boiling method may not be able to achieve as adequate a low-oxygen environment as in nodule. And oxygen may still be introduced when inoculating the bacteria under shaking culture conditions, which might be one of the reasons why *gfp* expression in the high-nitrogen, low-oxygen group was not as high as we expected.

##### Synthetic module

The RT-qPCR result indicated that our *pfa* gene biosynthetic cluster were successfully Transcribed. But the different transcription level between *pfa*1, *pfa*2 and *pfa*3 demonstrate a significant transcriptional decrease of large gene cluster, which might limit the efficiency of PUFA synthesis. We hypothesized that the incompatibility between weak *nuoA* promoter and large PUFA biosynthetic gene cluster might be the cause, and similar experiment proved our hypothesis(Figure14). Thus, *nifH* promoter from our original conception might have better performance in the same experiment. Because it also initiates the transcription of a nitrogenase cluster.

![](http://static.igem.wiki/teams/5300/source/engineering/14.png)

Figure14 The relative transcription level between *nuoA* promoter and *nifH* promoter measured by RT-qPCR. Student’s t-test, ns: no significant difference; \*, p-value < 0.05; \*\*, p-value%lt0.01; \*\*\*, p-value %lt 0.001.

As the gas chromatographic result show, it’s a pity that we did not identify the presence of DHA and EPA. Here is some reasons we supposed. First, the transcriptional reduction is the main cause, which lead to a bottleneck of *pfa*3 that encoding a lot of important enzymes in LC-PUFA synthetic pathway.

Second, the lack of incubation time also might be the cause. Because of the in vitro experiment, we could not unlimitedly extend the incubating time due to the limitation of nutrition. Thus, we only left a little time for rhizobia to translate PUFA synthetase and synthetize LC-PUFA.

Overall, the experiments of regulation module yielded results that were relatively fit our expectations, proving that the design of the regulation module was relatively successful. Although we did not obtained every ideal results of synthesis module, but few results shows the effectiveness of our design, which have a clear direction for iteration, improvement, and further validation. However, due to time constraints, we were unable to conduct more in-depth experiments.

## PHB deletion

The composite part composed of pJQ200SK, Up-Arm *phaC2* and Down-Arm *phaC2* has been engineered to delete the key gene *phaC2*, which is responsible for the synthesis of PHB in *Sinorhizobium fredii* CCBAU45436. The plasmid pJQ200SK can replicate in *E. coli* and can be conjugally transferred into rhizobia. The function of Up-Arm *phaC2* and Down-Arm *phaC2* is to facilitate homologous recombination with upstream and downstream of the *phaC2* gene. We first constructed pJQ200SK\_*phaC2*\_Deletion and utilized electron microscope to observe bacterial cells to assess its deletion efficiency. Ultimately, after implantation, we obtained the expected results.

### 1st iteration

**AIM:** The objective of the experiment is to delete *phaC2* to block the synthesis of PHB.

**DESIGN:** Through literature research, it was discovered that pJQ200SK can replicate in *E. coli* and can be conjugally transferred into rhizobia. Besides, it cannot replicate in rhizobia and is a suicide plasmid. The upstream and downstream DNA fragments of the target gene can be sequentially cloned into pJQ200SK to construct a homologous double-crossover suicide plasmid. This plasmid can be introduced into rhizobia by triparental mating, then DNA recombination between the plasmid and the genome is achieved under the action of the homologous recombination repair system. In this way, the target gene can be deleted. As a result, we attempted to use this method to delete *phaC2* in *Sinorhizobium fredii* CCBAU45436. Figure 15 shows the circuit we developed.

![](http://static.igem.wiki/teams/5300/source/engineering/15.png)

Figure15 Deletion\_*phaC2*\_plasmid

**BUILD:** Using PCR technology, we successfully obtained the uptream fragment(700bp) and downstream fragment(700bp) of *phaC2* from the genome of *Sinorhizobium fredii* CCBAU45436. To insert these two fragments into the multiple cloning sites of pJQ200SK, we utilized Gibson. The expression vector was transformed into *E. coli*(DH5α). The constructed genetic circuit was successfully developed, and its accuracy was confirmed through the implementation of colony PCR and sequencing techniques.

**TEST:** Using the triparental mating, we introduced it into *Sinorhizobium fredii* CCBAU45436. Utilizing Gm resistance carried on pJQ200SK, we screened for single recombinants. It's pity that we did not obtain any single recombinant.

**LEARN:** After literature research and analysis on the results, we suspect that the reason for the unsuccessful homologous recombination is the lack of sequence specificity in the upstream and downstream fragments.

### 2nd iteration

**DESIGN:** To increase sequence specificity, we re-selected the upstream(700bp) and downstream(700bp) fragments, which consist of genomic fragments upstream and downstream of *phaC2* and partial sequences of *phaC2*.

**BUILD:** Using PCR technology, we successfully obtained the uptream fragment(700bp) and downstream fragment(700bp) of *phaC2* from the genome of *Sinorhizobium fredii* CCBAU45436. To insert these two fragments into the multiple cloning sites of pJQ200SK, we utilized Gibson. The expression vector was transformed into *E. coli*(DH5α). The constructed genetic circuit was successfully developed, and its accuracy was confirmed through the implementation of colony PCR and sequencing techniques.

**TEST:** Using the triparental mating, we introduced it into *Sinorhizobium fredii* CCBAU45436. Utilizing Gm resistance carried on pJQ200SK, we screened for single recombinants. This time, we successfully obtained single recombinants(Figure 16).

![](http://static.igem.wiki/teams/5300/source/engineering/16.png)

Figure16 Single recombinants on solid medium.

Then we used a sucrose-killing gene (*sacB*), which encodes sucrose phosphorylase that can catalyze the hydrolysis of sucrose into glucose and fructose, and also polymerizes fructose into high molecular weight levan. The accumulation of high molecular weight levan has a potential toxic effect on cells, leading to cell death, and is applicable to various Gram-negative bacteria to screen the double recombinants. It's pity that we did not obtain any double recombinant.

**LEARN:** Although longer homologous regions can provide more sequence similarity, which helps the recognition of recombinase and promotes the occurrence of recombination events, overly long homologous regions may have some negative effects. For example, excessively long homologous regions might affect the stability of the vector. We suspect that the reason for the lack of double recombination is the unsuitable length of the upstream and downstream fragments.

### 3rd iteration

**DESIGN:** We adjusted the upstream(500bp) and downstream(500bp) fragments, which consist of genomic fragments upstream and downstream of *phaC2* and partial sequences of *phaC2*.

**BUILD:** Using PCR technology, we successfully obtained the uptream fragment(500bp) and downstream fragment(500bp) of *phaC2* from the genome of *Sinorhizobium fredii* CCBAU45436. To insert these two fragments into the multiple cloning sites of pJQ200SK, we utilized Gibson. The expression vector was transformed into *E. coli*(DH5α). The constructed genetic circuit was successfully developed, and its accuracy was confirmed through the implementation of colony PCR and sequencing techniques.

**TEST:** Using the triparental mating, we introduced it into *Sinorhizobium fredii* CCBAU45436. Utilizing Gm resistance carried on pJQ200SK, we screened for single recombinants. We successfully obtained single recombinants. Then we used a sucrose-killing gene to screen the double recombinants. This time, we successfully obtained double recombinants(Figure 17).

![](http://static.igem.wiki/teams/5300/source/engineering/17.png)

Figure17 Double recombinants on solid medium.

Then its accuracy was confirmed through the implementation of colony PCR(Figure18) and sequencing techniques(Figure19).

![](http://static.igem.wiki/teams/5300/source/engineering/18.png)

Figure18 Results of colony PCR.

![](http://static.igem.wiki/teams/5300/source/engineering/19.jpg)

Figure19 Sequencing result

**LEARN 3.0:** The pJQ200SK\_*phaC2*\_Deletion we designed definitely works. We successfully deleted the *phaC2* gene and explored factors that affect the effectiveness of the homologous recombination. Next, we conducted phenotypic experiments to compare the differences between the mutant and wild-type strains, testing the effect of inhibiting the PHB synthesis, which will be presented in the “Results” section.

## Suicide circuit

### 1st iteration

**DESIGN:** Through literature research, it was discovered that pJQ200SK can be introduced into rhizobia using triparental mating, and DNA recombination between the plasmid and the genome is achieved under the action of the homologous recombination repair system. In this way, the suicide circuit can be integrated into the genome of the rhizobia.

**BUILD:** Using the triparental mating, we introduced it into *Sinorhizobium fredii* CCBAU45436. Utilizing Gm resistance carried on pJQ200SK, we screened for single recombinants. We successfully obtained single recombinants.

**TEST:** We used a sucrose-killing gene to screen the double recombinants. We selected some positive colonies, cultured them overnight on a shaker at 28°C. Then spread the culture on solid media containing sucrose and NA, cultured them overnight at 28℃. It’s pity that we have faced microbial contamination (Figure20).

![](http://static.igem.wiki/teams/5300/source/engineering/20.jpg)

Figure20 Contaminated solid media

**LEARN:** After literature research and reflections on the experimental programme, we suspect that the reason for contamination is that rhizobia degraded a certain antibiotic in the culture media, allowing miscellaneous bacteria that do not have resistance to antibiotics we used to survive. Moreover, due to the introduction of a suicide pathway into rhizobia, miscellaneous bacteria have a competitive advantage over them, leading to contamination by miscellaneous bacteria.

### 2nd iteration

**AIM:** We suspect that the positive colonies on the plates used for single recombinants screening are contaminated with miscellaneous bacteria, so we want to further purify these positive colonies.

**DESIGN:** The streak plate method is a commonly used technique for the isolation and purification of microorganisms. It is easy to operate, with a low cost, to separate single colonies. The growth of colonies is easy to observe, and the isolated single colonies are convenient to store and less prone to contamination. For the above reasons, we have decided to use the streak plate method for further purification of positive colonies.

**TEST:** We picked positive colonies on the solid media used for screening single recombinants and streaked continuously on the solid media used for screening double recombinants. After incubating at 28°C for 5 days, we observed transparent colonies (Figure21). Then we identified these colonies as double recombinants using colony PCR(Figure22).

![](http://static.igem.wiki/teams/5300/source/engineering/21.jpg)

Figure21 Transparent colonies

![](http://static.igem.wiki/teams/5300/source/engineering/22.jpg)

Figure22 Electropherogram Marker, double recombinants(lane4&5), WT (lane10)

**LEARN 2.0:** Our purification method definitely works. We have successfully purified single recombinants and screened for double recombinants. Next, we conducted stage verification to test the function of our suicide circuit, which will be presented in the “Results” section.

# Dry lab engineering

## Software

### 1st iteration: Software Construction

**DESIGN:** At first, we wanted to design simple software of retrieving pathways when input substrate. So, we intended to create a database, where we could store and fetch data.

**BUILD:** We chose MATLAB App designer to create software interface and chose MySQL to store and fetch data. We stored selected data in MySQL, and then wrote code on MATLAB App designer, linking MySQL to read the data.

![](http://static.igem.wiki/teams/5300/source/engineering/23.png)

Figure23 Picture of using MySQL to store data

**TEST:** After constant debugging, we finally made it. When we entered a substrate, it would pop up all the relevant pathways we had stored in our MySQL database.

![](http://static.igem.wiki/teams/5300/source/engineering/24.jpg)

Figure24 Picture of the initial software interface

**LEARN:** When using software, the input substrate could be directly retrieved to the relevant pathways, which made the call of data very convenient.

### 2nd iteration: Changing Databases

**AIM:** For the sake of users, we realized that many users do not have a MySQL database configured on their computers, and it might be very inconvenient for them to connect to the database. We hoped that our software was friendly for non-professional users.

**DESIGN:** We finally selected EXCEL as our database to store data. Because EXCEL is a widely used tool, and it is very convenient to fetch data from EXCEL.

**BUILD:** We changed the database to EXCEL by adjusting the code.

**TEST:** We made the polished software available to other members of the team and found that it could be used by much wider users than MySQL.

**LEARN:** If we want to build software that can be used widely, it's better to use configurations that most people are using.

### 3rd iteration: Configuration Files of Pathways

**AIM:** In addition to displaying the relevant pathways retrieved, we wanted to display the concentrations of each product in the pathway in real time, to investigate the changes of the products we focused on.

**DESIGN:** We chose SSH keys as a linker between Computer terminal and files of pathways hosted on GitLab.

**BUILD:** We configured the SSH public key, posted the relevant files of pathways on GitLab, and wrote the code to use SSH to clone the files into the main folder of the computer for calling and running.

**TEST:** After continuous debugging, we successfully realized the process of inputting substrates - retrieving relevant pathways - selecting pathways - running pathway files.

**LEARN:** With a real-time visualization of product concentrations, we hoped to make the pathway investigations more easily for other groups.

### 4th iteration: Modification of Concentration

**AIM:** As we delved deeper into usability, we realized that conducting quantitative analyses was constrained by the inability to modify substrate concentrations. Therefore, we dedicated significant efforts to enable users to adjust substrate concentrations, allowing for the generation of concentration curves for various products.

**DESIGN:** We planned to read data from EXCEL when inputting substrates to display the relevant pathways. Subsequently, the software matched the pathway name to files cloned via SSH. Within the file, the software searched for the name of the substrate we input, allowing us to modify the desired concentration values. Once we had made these changes, the software proceeded to run the modified file. After completion, the substrate values will be restored to their initial states.

**BUILD:** We accomplished the aim by writing code.

**TEST:** The result was satisfactory. For more information of our final results, please visit our [wiki page](https://2024.igem.wiki/cau-china/software).

**LEARN:** Please visit the discussion of our [wiki page](https://2024.igem.wiki/cau-china/software).

### 5th iteration: Software beautification and error tips

**AIM:** We wanted to make the users gain great experience.

**DESIGN:** We decided to beautify the interface and add some feedback services.

**BUILD:** We designed the software logo and interface pictures by ourselves, then beautified the curve graph for the files we collected and added legends. Eventually, we added the error program. We ensured that the design is not only visually appealing but also user-friendly, including the addition of legends to each graph that were omitted in the original version.

**TEST:** We tested the error-reporting program to make sure it worked.

![](http://static.igem.wiki/teams/5300/source/engineering/25.png)

Figure25 Picture of the beautified software

![](http://static.igem.wiki/teams/5300/source/engineering/26.png)

Figure26 Picture of error tip

**LEARN:** It seems that our software has great potential for application. Currently, we are consistently expanding our database to include pathways related to immune regulation, gene expression, and cell differentiation, building upon our foundational metabolic pathways. This expansion aims to create a more comprehensive and powerful resource for our users.

## Nodule Formation Model

### 1st iteration: 2D cellular automata

**DESIGN:** Since our project focuses on nodules, we want to predict the formation of nodules and provide some guidance to the project. The growth of Sinorhizobium and the formation of nodules follow a definite pattern which has a strong periodicity. We initially used 2D cellular automata to simulate the changes of the nodule section. Through literature research, we got the basic understanding of the mechanism.

**BUILD:** According to the literature, we made some assumptions based on the rhizomatous mechanism to simplify the model. After that, we set up the four states of the cellular automata, including empty, root, bacteria and sheath. Finally, we set up the Moore neighborhoods and the rules for state transitions.

**TEST:** We finished coding and visualizing the results using Python. The code establishes a 2D coordinate system and implements the logic of the cellular automata. The results provide a good picture of the dynamics of the 2D cross-section during nodule formation.

**LEARN:** By observing the results of the run, we found that 2D cellular automata cannot well simulate the rhizomes with 3D structures. For example, Sinorhizobium interaction in the longitudinal direction is difficult to represent, and we can't know its formation mechanism in the z-axis. Also, since we want to calculate DHA production, it’s necessary to quantify the amount of Sinorhizobium in nodules, which needs further improvement.

### 2nd iteration: 3D cellular automata 1.0

**AIM:** We need to simulate the mechanism of nodules formation in z-axis and quantify the changes in the number of Sinorhizobium in nodules. It is the basis for calculating the DHA yield.

**DESIGN:** Considering that we want to complete the 3D modeling, we change the original basis to 3D cellular automata and change the whole system to 3D coordinate system.

**BUILD:** We kept the four states of the cellular automata and changed the neighborhoods to 3D Moore neighborhoods. In addition, we transformed the rules to interact in 3D.

**TEST:** We used python to code and visualize the program. The code establishes a 3D coordinate system and implements the logic of the cellular automata. The result is a good representation of the dynamics of nodules formation in three dimensions.

**LEARN:** 3D cellular automata can simulate the formation of nodules very well. We showed the simulation results to our instructor Prof. Jiao, who said that the existing rules for the formation of rhizomes are simple and cannot simulate the actual situation of growth, so he recommended us literature to read and modify the rules. In addition, we found that the results are not aesthetically pleasing and need to be improved.

![](http://static.igem.wiki/teams/5300/source/engineering/27.png)

Figure27 Result of 3D cellular automata 1.0

### 3rd iteration: 3D cellular automata 2.0

**AIM:** Set up more detailed 3D cellular automata rules based on the latest literature on nodules' formation. Beautify the result. Make curves of the number of each state versus iterations for quantitative calculations.

**DESIGN:** We augmented the states in the 3D cellular automata strictly according to the mechanism of nodule' formation demonstrated in the literature and formulated new state transition rules.

**BUILD:** We set up six states of the cellular automata, including empty, root, bacteria, attached bacteria, infection thread, nodule, \(N\_{2}\) fixing nodule. In addition, we changed the neighborhoods to 3D Moore neighborhoods. After that, we developed new and reliable rules and transformed them into 3D interactions.

**TEST:** We wrote and visualized the program in Python. The code establishes a 3D coordinate system and implements the logic of the cellular automata. The results have been embellished by choosing different colors to represent different states.

**LEARN:** 3D cellular automat 2.0 can simulate the actual way of nodule formation very well and gives a great reference for the whole project. We obtained the variation of the number of states with the number of iterations, which facilitates subsequent quantitative calculations, such as the prediction of DHA yield.

![](http://static.igem.wiki/teams/5300/source/engineering/28.png)

Figure28 Result of 3D cellular automata 2.0

## Nitrogen Regulatory Protein Docking Model

### 1st iteration: AlphaFold3 model

**DESIGN:** The production of DHA is regulated by nitrogen. Although in *Rhizobium etli*, NtrC protein is proven to be feasible according to the literature, we need to know whether the parts we select are suitable for *S. fredii* CCBAU45436. Furthermore, we can verify the broad-spectrum nature of nitrogen regulation in rhizobia. In short, the essence of the problem is to simulate protein-DNA docking, where the DNA sequence is known, but the structure of NtrC protein needs to be predicted.

**BUILD:** We chose Alphafold3 to predict the structure of the NtrC protein in both species, and then continued to predict the protein-DNA docking using Alphafold3. Afterwards, we used PyMol to visualize and analyze the interactions of the bases and amino acid residues involved.

**TEST:** Alphafold predicts protein structure with high confidence. However, its prediction of protein-DNA docking is unsatisfactory: the docking of NtrC with the *Rhizobium etli* *glnK/amtB* operon is the opposite of what has been demonstrated in literature. We completed the docking results visualization and parameter analysis using PyMol.

**LEARN:** We assumed that Alphafold3 has a limition to predict protein-DNA docking, as it is unable to complete the validation of the conclusions already made in the literature. Further, we decided to replace Alphafold3 with a model specialized in protein-DNA docking.

![](http://static.igem.wiki/teams/5300/source/engineering/29.jpg)

Figure29 The Alphafold3 docking result of NtrC protein and *glnK/amtB* operon in *Rhizobium etli*

### 2nd iteration: HDOCK model

**AIM:** Based on the prediction that the model can validate the findings of the literature on *Rhizobium etli*, we will predict whether the same regulatory mechanism can be utilized in *S. fredii* CCBAU45436.

**DESIGN:** After reviewing a large amount of literature, we decided to use HDOCK to predict protein-DNA docking, which combines template-based and free docking, allows for efficient prediction of protein-DNA binding.

**BUILD:** We imported the NtrC protein structure.pdb file predicted by Alphafold3 and the *glnK/amtB* operon sequence into the HDOCK model for prediction. We completed the docking results visualization and parameter analysis using PyMol.

**TEST:** The confidence score of docking given by HDOCK validated the findings in the literature and was found to be higher in the predicted *S. fredii* CCBAU45436. In addition, we determined the bases and residues at the binding site, the length of the hydrogen bond, the length of the protein-DNA binding, and the length of the interactions by PyMol.

**LEARN:** HDOCK is an excellent predictor of protein-DNA docking and confirms the literature and our conjecture. In our chassis *S. fredii* CCBAU45436, the NtrC protein is homologous to that in *Rhizobium etli* and can perform the same function. This greatly enhances the confidence of our project to regulate DHA synthesis with nitrogen.

![](http://static.igem.wiki/teams/5300/source/engineering/30.png)

Figure30 The HDOCK docking result of NtrC protein and *glnK/amtB* operon in *S. fredii* CCBAU45436

## DHA Yield Prediction Model

### 1st iteration: Flux Balance Analysis (FBA) Model

**DESIGN:** Initially, we focused on constructing a model that captured a static state of the metabolic system, not accounting for time-dependent changes. To ensure data accuracy and analyze the system’s steady-state, we chose Flux Balance Analysis (FBA) as our modeling approach. FBA allows us to simulate metabolic fluxes based on mass balance constraints without considering dynamics over time.

**BUILD:** The FBA model was developed and implemented to simulate the metabolic pathways under static conditions. This provided a foundation for understanding how metabolic fluxes were distributed across various pathways in the system.

**TEST:** We ran simulations with the FBA model to predict the metabolic fluxes under given conditions. These results were useful for identifying potential bottlenecks and optimal flux distributions within the metabolic network.

**LEARN:** While the FBA model provided insights into the static distribution of metabolic fluxes, it became clear that real biological systems exhibit dynamic behavior over time, which could not be captured by this model. As a result, we concluded that to better simulate the system, we needed to account for time-dependent changes, leading us to the next iteration.

### 2nd iteration: Ordinary Differential Equations (ODE) Model

**AIM:** To incorporate time-dependent dynamics and better simulate biological processes, we aimed to build a model that could reflect changes in the system over time.

**DESIGN:** We designed an Ordinary Differential Equations (ODE) model that would enable us to track the time-course behavior of key metabolites and enzymes. The ODE model provided a framework for understanding how the concentrations of these components evolve over time based on reaction rates and other dynamic factors.

**BUILD:** The ODE model was constructed and implemented, with time-dependent equations representing the interactions between metabolites and enzymes. This allowed us to simulate the temporal changes in the system under various initial conditions.

**TEST:** We ran simulations using the ODE model and performed sensitivity analysis to evaluate how changes in various parameters affected the system’s behavior over time. These tests revealed key insights into which parameters had the most significant impact on the model's outputs.

![](http://static.igem.wiki/teams/5300/source/engineering/31.jpg)

Fig31 Initial results of the ODE model

**LEARN:** Sensitivity analysis showed that the activity of PUFA synthase had a major effect on the model’s results. It was also observed that PUFA synthase activity is regulated by nitrogen availability, indicating that nitrogen dynamics play a critical role in the system. This prompted the need to incorporate nitrogen absorption into the model.

### 3rd iteration: Nitrogen Absorption Model

**AIM:** The next goal was to incorporate the influence of nitrogen into the model to better capture the regulation of PUFA synthase and its effects on the metabolic pathway.

**DESIGN:** A Nitrogen Absorption Model was designed to account for the effects of nitrogen availability on PUFA synthase activity. This model would enable us to simulate how fluctuations in nitrogen levels impact the system’s metabolic processes.

**BUILD:** The Nitrogen Absorption Model was integrated with the existing ODE framework. We included equations that represented nitrogen uptake and its regulatory effect on PUFA synthase, ensuring that the interactions between nitrogen availability and enzyme activity were accurately reflected.

**TEST:** Simulations were conducted to assess how varying levels of nitrogen affected the system over time. These tests provided a deeper understanding of the relationship between nitrogen absorption and DHA yield.

**LEARN:** The Nitrogen Absorption Model successfully demonstrated the critical role of nitrogen in DHA yield prediction. The integrated model now offers a more accurate depiction of the biological system, showing how nitrogen availability influences metabolic fluxes and fatty acid synthesis over time.

## Suicide Circuit Model

### 1st iteration: Toxin Expression Model

**DESIGN:** In the initial stages, we referenced Part: BBa\_K3512042. The toxin expression equations for our suicide circuit were listed on the basis of their differential equations. We didn’t take the influence of newly generated proteins towards the production of proteins in to consideration.

**BUILD:** We listed basic differential equations according to the interactions in the circuit. And ran the model at MATLAB.

**TEST:** At the beginning, there are always errors when we ran the model, and sometimes the solution couldn’t be figured out. It took a long time to get the solution, and the resulting curve was unusual.

**LEARN:** By looking up literature and analyzing the material interactions repeatedly, we found our logical flaws in the equations. The equations were not accurate enough because the relationships between substance interaction were not understood correctly.

### 2nd iteration: Suicide Circuit Model

**AIM:** We wanted to rebuild the interaction lines and enhance the differential equations, thus gaining a more accurate result.

**DESIGN:** We reformulated the differential equations with deeper understanding of the circuit, and made innovative modifications to the equations that were more realistic: adding a condition that the tendency of newly generated proteins slow down the production of proteins.

**BUILD:** We incorporated a mechanism where the accumulation of newly generated proteins negatively regulates their own production, featured two response-regulated promoters, and included interactions between Cas12K and sg-mCherry, as well as the mutual binding dynamics of VapC and VapB.

**TEST:** We ran the model at MATLAB. The result was as expected. It was observed that only in a high-nitrogen and high-oxygen environment does the net expression of toxins increase significantly, leading to the suicide of Sinorhizobium.

![](http://static.igem.wiki/teams/5300/source/engineering/32.png)

Figure32 Expression content plot of toxins in different cases

**LEARN:** It can be concluded that the suicide pathway was safe and feasible in a single invariant environment.

### 3rd iteration: Whole Process of Plant Growth Model

**AIM:** The growth environment of Sinorhizobium is variable, so on the basis of a single invariant environment, we wanted to know whether the suicide circuit was safe during the whole growth process. Beside that, we wanted our result more intuitive and visually appealing.

**DESIGN:** We set up different differential equations according to the environmental changes during Sinorhizobium growth. We adjusted the colors of the curves and refined the legends to improve the visual presentation.

**TEST:** The result was satisfactory. The amount of toxin in the early stage wasn’t high enough. Once the nodules escaped, the toxin content quickly increased, leading to the death of Sinorhizobium, thus preventing gene pollution and other issues. At the same time, the presentation of the results was pretty.

![](http://static.igem.wiki/teams/5300/source/engineering/33.png)

Figure33 Expression content plot of toxins in different cases(plus)

![](http://static.igem.wiki/teams/5300/source/model/newfigure24.png)

Figure34 Expression content plot of toxins in whole process

**LEARN:** We concluded that our suicide circuit is safe and feasible. Through iterative modifications and refinements of these equations, we developed more complex and rigorous parameterized differential equations. This comprehensive modeling effectively simulates the full process of the suicide circuit's operational mechanisms.

{% endblock %}
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{% extends "layout.html" %}
{% block title %}Team{% endblock %}
{% block lead %}On this page you can introduce your team members, instructors, and advisors.{% endblock %}
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![](https://static.igem.wiki/teams/5300/source/experiment/top.png)

[Preparation of basic experimental material](#1)

[Molecular Biology Experiment](#2)

[Microbiology Experiment](#3)

[Plant Experiment](#4)

[Others](#5)

# Preparation of Basic Experimental Material

### 1. LB

#### Materials:

* Tryptone
* Yeast extract
* NaCl AR
* dH2O
* Conical flask
* Microwave
* Magnetic stirrer and rotor

#### Protocol:

1. Weigh reagents in the following proportions:

   | Substance | Concentration |
   | --- | --- |
   | Tryptone | 10 g/L |
   | Yeast extract | 5 g/L |
   | NaCl | 10 g/L |
2. Deposit them all into the flask.
3. Add distilled water to the expected volume.
4. Stir the mix until the powders are completely dissolved.
5. Autoclave for 20 minutes at 121°C.
6. Store at 4°C.

### 2. LB Agar

#### Materials:

* Tryptone
* Yeast extract
* NaCl AR
* Agar
* dH2O
* Conical flask
* Microwave
* Magnetic stirrer and rotor

#### Protocol:

1. Weigh reagents in the following proportions:

   | Substance | Concentration |
   | --- | --- |
   | Tryptone | 10 g/L |
   | Yeast extract | 5 g/L |
   | NaCl | 10 g/L |
2. Deposit them all into the flask.
3. Add distilled water to the expected volume.
4. Stir the mix until the powders are completely dissolved.
5. Add 1.5% weight agar into the flask.
6. Autoclave for 20 minutes at 121°C.
7. Store at 4°C.

### 3. Preparation of LB-Antibiotics

* If the medium does not contain agar: Add one thousandth of a volume of antibiotic solution, mix thoroughly.
* If the medium does contain agar: When the temperature is reduced to approximately 45°C, add one thousandth of a volume of antibiotic solution, then mix it thoroughly. After antibiotics are added, it should not be heated again.

### 4. Preparation of Agarose Gel

#### Materials:

* Agarose
* 1x TAE
* Graduated cylinder
* Conical flask
* Pipettor and pipette
* Microwave
* 10,000x nucleic acid dye

#### Protocol:

1. Add the exact weight of agarose to the measuring cylinder to ensure the correct concentration.
2. Add 1x TAE of the required volume.
3. Heat the solution in the microwave until it boils.
4. Take the cylinder out and let it cool down until it can be touched by hands.
5. Add nucleic acid dye that is 1/10,000 of the total volume of the system.
6. Pour into electrophoresis box, place the comb, and wait until it solidifies.
7. Take out the comb carefully.

### 5. TY Medium

#### Materials:

* Tryptone
* Yeast extract
* CaCl2 AR
* dH2O
* Conical flask
* Microwave
* Magnetic stirrer and rotor

#### Protocol:

| Substance | Concentration |
| --- | --- |
| Tryptone | 5 g/L |
| Yeast extract | 3 g/L |
| CaCl2 | 0.6 g/L |

1. Deposit them all into the flask.
2. Add distilled water to the expected volume.
3. Stir the mix until the powders are completely dissolved.
4. Autoclave for 20 minutes at 121°C.
5. Store at 4°C.

### 6. TY Agar

#### Materials:

* Tryptone
* Yeast extract
* CaCl2 AR
* Agar
* dH2O
* Conical flask
* Microwave
* Magnetic stirrer and rotor

#### Protocol:

| Substance | Concentration |
| --- | --- |
| Tryptone | 5 g/L |
| Yeast extract | 3 g/L |
| CaCl2 | 0.6 g/L |

1. Deposit them all into the flask.
2. Add distilled water to the expected volume.
3. Stir the mix until the powders are completely dissolved.
4. Add 2% weight agar into the flask.
5. Autoclave for 20 minutes at 121°C.
6. Store at 4°C.

If needed, 5% sucrose can be added to TY or TY agar.

### 7. 5xM9 Medium

#### Materials:

* Na2HPO4
* KH2PO4
* NaCl AR
* CaCl2 AR
* MgSO4·7H2O
* NH4Cl
* Glucose
* dH2O
* Conical flask
* Microwave
* Magnetic stirrer and rotor

#### Protocol:

1. Weigh reagents in the following proportions for the 5xM9 basic salt solution:

   | Substance | Concentration |
   | --- | --- |
   | Na2HPO4 | 33.9 g/L |
   | KH2PO4 | 15.0 g/L |
   | NaCl | 2.5 g/L |
2. Heat to dissolve, make up to 400 ml, high-temperature sterilize, and separately prepare the following three solvents.
3. Weigh reagents in the following proportions for 1M MgSO4·7H2O:

   | Substance | Concentration |
   | --- | --- |
   | MgSO4·7H2O | 246.5 g/L |
4. Weigh reagents in the following proportions for 1M CaCl2:

   | Substance | Concentration |
   | --- | --- |
   | CaCl2 | 111 g/L |
5. Weigh reagents in the following proportions for 100xNH4Cl:

   | Substance | Concentration |
   | --- | --- |
   | NH4Cl | 100 g/L |
6. Prepare the three solvents separately and high-temperature sterilize.
7. Weigh reagents in the following proportions for 20% Glucose Solution:

   | Substance | Concentration |
   | --- | --- |
   | Glucose | 200 g/L |
8. Sterilize by filter membrane.
9. Take 100 ml of 5xM9 basic salt solution, add 10 ml of 20% Glucose Solution, 1 ml of 1M MgSO4·7H2O, and 50 µL of CaCl2 solution, and make up to 500 ml.
10. Based on the concentration gradient set, add the corresponding amount of 100xNH4Cl as required.

# Molecular Biology Experiment

### 1. Genome Extraction

#### Materials:

1. Centrifuge tubes (1.5 mL & 2.0 mL)
2. G+ Bacteria Genomic DNA Kit (Zomanbio) (Lysozyme (100 mg/mL), cell suspension solution, Buffer A, lysis solution S, Buffer B, Buffer C, Washing Solution W2, Elution Buffer TE, Elution Buffer Columns, Collection Tube)
3. RNase A (10 mg/mL) (Zomanbio)
4. ddH2O
5. Ethanol
6. Centrifugal machine
7. Vortex machine
8. Water bath

#### Protocol:

1. Centrifugate 1-5 mL bacteria solution for 1 min at 13,400xg to retain the pellet.
2. Add 500 μl cell suspension solution (with Lysozyme added) to the bacterial pellet, vortex until the pellet is re-suspended. Incubate at 37℃ for 30 minutes, inverting every 5-10 minutes. Centrifuge at 13,400xg for 2 minutes and thoroughly remove the supernatant.
3. Add 225 μl Buffer A and mix thoroughly.
4. Add 6 μl RNase A (10 mg/mL) solution, vortex for 5 seconds and mix well.
5. Add 25 μl lysis solution S and mix thoroughly. Incubate at 56℃ for 20 minutes, inverting 1-2 times during this period.
6. Add 250 μl Buffer B, vortex for 5 seconds and mix thoroughly.
7. Add 250 μl ethanol, vortex for 15 seconds. Transfer the mixture to the column and place the column in the collection tube.
8. Centrifuge at 13,400xg for 30 seconds, discard the supernatant. Place the absorption column in the collection tube.
9. Add 500 μl Buffer C, centrifuge at 13,400xg for 30 seconds, discard the supernatant, and place the column back into the collection tube.
10. Add 600 μl Washing Solution W2 (with ethanol added), centrifuge at 13,400xg for 30 seconds, discard the supernatant, and place the column back into the collection tube.
11. Add an additional 500 μl Washing Solution W2 (with ethanol added), centrifuge at 13,400xg for 30 seconds, discard the supernatant, and place the column back into the collection tube.
12. Centrifuge for 2 minutes at 13,400xg. Open the column and let it sit at room temperature for a few minutes.
13. Place the column in a new 1.5 mL centrifuge tube, add 100-200 μl elution buffer TE, and let it sit at room temperature for 2-5 minutes.
14. Centrifuge for 2 minutes at 13,400xg. Transfer the liquid in the collection tube to the column.
15. Dispose of the column and store the eluate at -20℃.

### 2. PCR

#### 2.1 General PCR

**Goal:** Amplify a gene or add homology arms to a sequence.

#### Materials:

* PCR tubes (200 μl)
* GoldenStar T6 Super PCR Mix (1.1x)
* Forward primer (10 μM)
* Reverse primer (10 μM)
* Template
* Thermocycler

#### Protocol:

- Add in a PCR tube:

  | Substance | Volume (system 25μl/50μl) |
  | --- | --- |
  | Golden Mix | (23-x) μl/(46-x) μl |
  | Forward primer (10 μM) | 1 μl/2 μl |
  | Reverse primer (10 μM) | 1 μl/2 μl |
  | Template | x μl (depends) |
- Place the tube in the thermocycler and start corresponding program.
- After the sample temperature decreases to 4℃, store the result at -20℃.

#### 2.2 Fusion PCR

**Goal:** Connect two DNA fragments containing homologous arms together.

#### Materials:

1. PCR tubes (200 μl)
2. GoldenStar T6 Super PCR Mix (1.1x)
3. Template (containing homologous arms)
4. Forward primer (10 μM)
5. Reverse primer (10 μM)
6. Thermocycler

#### Protocol:

- Add in a PCR tube:

  | Substance | Volume (system 25μl/50μl) |
  | --- | --- |
  | Golden Mix | (23-x-y) μl/(46-x-y) μl |
  | Template1 | x μl (depends) |
  | Template2 | y μl (depends) |
- Add 1 μl/2 μl of each of the two primers.
- Place the tube in the thermocycler and start corresponding program.
- After the sample temperature decreases to 4℃, store the result at -20℃.

#### 2.3 Colony PCR

**Goal:** To determine whether a certain sequence exists in the thallus.

#### Materials:

1. PCR tubes (200 μl)
2. 2 x Taq Master Mix (dye) / 2 x T5 Super PCR Mix (Basic)
3. Forward primer (10 μM)
4. Reverse primer (10 μM)
5. ddH2O
6. Thermocycler

#### Protocol:

1. Add in a PCR tube:

   | Substance | Volume (system 25μl) |
   | --- | --- |
   | 2 x Taq Master Mix (dye) / 2 x T5 Super PCR Mix (Basic) | 12.5 μl |
   | Forward primer (10 μM) | 1 μl |
   | Reverse primer (10 μM) | 1 μl |
   | ddH2O | 10.5 μl |
2. Usually, we configure more premix than the required number of systems in a 1.5 mL centrifuge tube, then separate them into PCR tubes.
3. Place the tube in the thermocycler and start corresponding program.
4. After the sample temperature decreases to 4℃, stop the program and use the results for AGE detection.

### Plasmid Extraction

### 3.1 Zomanbio

#### Materials:

* Centrifuge tubes (1.5 mL & 2.0 mL)
* Fast Plasmid Miniprep Kit (Zomanbio) (RNase A (10 mg/mL), Solution K1, Solution K2, Solution K3, Deproteinized solution W1, Buffer V2, Washing Solution W2, Elution Buffer TE, Elution Buffer Columns, Collection Tube)
* Centrifugal machine
* Vortex machine

#### Protocol:

1. Centrifugate 1-5 mL bacteria solution 1 min at 13,400 x g, retain precipitation.
2. Add 250 μl Buffer K1 (RNase added) in the bacteria precipitation, vortex it until the thallus is resuspended.
3. Add 250 μl buffer K2, gently mix it until the liquid looks clear.
4. Add 350 μl buffer K3, immediately mix it gently.
5. Centrifugate the tube 2 min at 13,400xg, transfer the supernatant to column and put it in the collection tube, centrifugate 30-60 s at 13,400xg. Discard supernatant, replace the column into the collection tube.
6. If the bacteria is *E. coli* BL21 (DE3) or Pseudomonas fluorescens 2P24, add 500 μl Deproteinized solution W1 in column and centrifugate 30-60 s at 13,400xg. If the bacteria is *E. coli* DH5 α, skip this step.
7. Add 500-600 μl Washing Solution W2 (ethanol added) in column and centrifugate 20 s at 13,400xg. Repeat this step.
8. Centrifugate 1 min at 13,400xg.
9. Put the column in a new centrifuge tube, add 80-150 μl elution buffer TE. Place it for 1 min.
10. Centrifugate 1 min at 13,400xg. Discard the column and store the result at -20℃.

### 3.2 TransGen

#### Materials:

* Centrifuge tubes (1.5 mL & 2.0 mL)
* EasyPure Plasmid Miniprep Kit (TransGen) (RNase A (10 mg/mL), Resuspension Buffer (RB), Lysis Buffer (LB), Neutralization Buffer (NB), Wash Buffer (WB), Elution Buffer (EB), Mini-Plasmid Spin Column with Collection Tubes)
* Centrifugal machine
* Vortex machine

#### Protocol:

1. Before use, RNase A was added to RB and stored at 2-8℃. Add different volumes of 100% ethanol to WB.

| LB Media Volume | RB (μl) | LB (μl) | NB (μl) |
| --- | --- | --- | --- |
| 5 mL | 250 | 250 | 350 |
| 5-10 mL | 500 | 500 | 700 |
| 10-15 mL | 750 | 750 | 1,050 |
| 15-20 mL | 1,000 | 1,000 | 1,400 |

2. Take the overnight culture, centrifuge at 10,000xg for 1 minute, and remove the supernatant.
3. According to the table, add colorless solution RB (containing RNase A), vortex to resuspend the bacterial pellet, ensuring no small clumps remain.
4. According to the table, add blue solution LB, gently invert 4-6 times to mix, ensuring complete lysis of the bacteria, forming a blue and clear solution. The color change from semi-transparent to clear blue indicates complete lysis.
5. According to the table, add yellow solution NB, gently mix 5-6 times (the color change from blue to yellow indicates complete mixing and neutralization), until a compact yellow precipitate forms, then let it stand at room temperature for 2 min.
6. Centrifuge at 12,000xg for 5 min, carefully transfer the supernatant to a centrifuge column. Centrifuge at 12,000xg for 1 min, discard the flow-through.
7. Add 650 μl solution WB, centrifuge at 12,000xg for 1 min, and discard the flow-through.
8. Centrifuge at 12,000xg for 1-2 min to thoroughly remove residual WB.
9. Place the centrifuge column in a clean centrifuge tube, add 20 μl EB to the center of the column, and let it stand at room temperature for 1 min. (Pre-warming EB in a 60-70°C water bath improves the outcome).
10. Centrifuge at 10,000xg for 1 min to elute the DNA, and store the eluted DNA at -20°C.

### 3.3 Mei5bio

#### Materials:

* Centrifuge tubes (1.5 mL & 2.0 mL)
* M5 HiPer Multi-color Plasmid Miniprep Kit (Mei5bio) (RNase A (10 mg/mL), Solution Ⅰ, Solution Ⅱ, Solution Ⅲ, Buffer WB1 (with anhydrous ethanol added), Buffer WB2 (with anhydrous ethanol added), Buffer BL, Buffer EB, Washing Solution W2, Elution Buffer TE, Elution Buffer Columns, Collection Tube)
* Centrifugal machine
* Vortex machine

#### Protocol:

1. Column Equilibration: Add 400 μL of Buffer BL to the centrifugal adsorption column, let it stand for 1 minute, centrifuge at 12,000 rpm at room temperature for 1 minute, discard the waste liquid in the collection tube, and place the centrifugal adsorption column back into the collection tube.
2. Take 1-5 mL of overnight culture, centrifuge at 12,000 rpm at room temperature for 1 minute, and remove the supernatant as completely as possible.
3. Add 250 μL of Solution I, vortex or pipette thoroughly to resuspend the bacteria evenly, resulting in a uniformly turbid brown-red color.
4. Add 250 μL of Solution II, gently invert to mix well, allowing the bacteria to fully lyse until the solution becomes clear and viscous purple-red.
5. Add 350 μL of Solution III, immediately and gently invert to mix, a precipitate with red and yellow layers will be visible, continue mixing until it turns completely yellow, let it stand at room temperature for 2 min, then centrifuge at 12,000 rpm for 5 min.
6. Carefully transfer the supernatant to the centrifugal adsorption column, let it stand for 2 min to allow the plasmid to bind fully with the silica membrane of the column. Centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate in the collection tube.
7. Add 500 μL of Buffer WB2, centrifuge at 12,000 rpm at room temperature for 30 seconds, and discard the filtrate in the collection tube. Repeat this step once.
8. Centrifuge at 12,000 rpm at room temperature for 2 min to dry the residual liquid.
9. Place the centrifugal adsorption column in a new 1.5 mL plastic centrifuge tube (provided by the user), add 50-100 μL of elution buffer EB, let it stand at room temperature for 2 min.
10. Centrifuge at 12,000 rpm for 1 minute, and the solution at the bottom of the centrifuge tube is the plasmid DNA. Store the eluted DNA at -20℃.

### Plasmid Digestion

**Goal:** To cleave a vector using restriction enzymes to prepare them for ligation.

### 4.1 pBBR1MCS-2, *EcoR*Ⅰ + XmaⅠ

#### Materials:

* *Eco*RⅠ
* XmaⅠ
* pBBR1MCS-2
* rCutSmart Buffer (10x)
* ddH2O
* 200 μL centrifuge tube
* Water bath at 37℃
* Water bath at 65℃

#### Protocol:

| Substance | Volume |
| --- | --- |
| EcoRⅠ | 0.1 μl |
| XmaⅠ | 0.1 μl |
| pBBR1MCS-2 | x μl (depends) |
| rCutSmart Buffer (10x) | 1 μl |
| ddH2O | (8.8-x) μl |

1. Keep warm in a water bath at 37℃ for 1 hour.
2. Keep warm in a water bath at 65℃ for 20 minutes.
3. Correct bands were extracted after electrophoresis.

### 4.2 pJQ200SK, SmaⅠ

#### Materials:

* SmaⅠ
* pJQ200SK
* 10X QuickCut Green Buffer
* ddH2O
* 200 μL centrifuge tube
* Water bath at 37℃
* Water bath at 80℃

#### Protocol:

| Substance | Volume |
| --- | --- |
| SmaⅠ | 1 μl |
| pJQ200SK | x μl (depends) |
| 10X QuickCut Green Buffer | 1 μl |
| ddH2O | (8-x) μl |

1. Keep warm in a water bath at 37℃ for 1 hour.
2. Keep warm in a water bath at 80℃ for 15 minutes.
3. Correct bands were extracted after electrophoresis.

### Agarose Gel Electrophoresis (AGE)

**Goal:** Separating DNA bands according to their length, and estimating the length and DNA concentration of each band. When digested products or recombinant products are subjected to electrophoresis, care should be taken to use the original plasmids for control.

#### Materials:

* Ready-made agarose gel
* DNA sample
* 10 x loading buffer (dye)
* DNA Ladder/Marker
* 1 x TAE
* Electrophoresis tank

#### Protocol:

1. Place the gel on the gel tray within the electrophoresis system, make sure the wells are located at the negative electrode.
2. Mix loading buffer with the DNA sample if the sample is not coloured. Usually, the loading buffer has one tenth the volume of the sample, but the volume should not be less than 0.3 μl.
3. Load either the DNA ladder or the samples within the wells. Make sure that the sample or ladder sinks to the bottom of the well. The volume should be between 3 μl to 5 μl, depending on the well's capacity.
4. Run the gel for 20-40 minutes, depending on the situation. Ensure the voltage is set to 100-150 V.

### DNA Extraction from Agarose Gel

#### Materials:

* Centrifuge tubes (1.5 mL)
* EasyPure Quick Gel Extraction Kit (TransGen) (Wash Buffer WB, Elution Buffer EB, Gel Solubilization Buffer GSB, Gel Spin Columns with Collection Tubes)
* ddH2O
* Centrifugal machine
* Vortex machine
* Water bath

#### Protocol:

1. Carefully cut the target band on agarose gel and put it in a centrifuge tube.
2. Add 600 μl GSB. Heat it at 55℃ using a water bath for 6-10 minutes. Mix intermittently to ensure the gel is completely melted.
3. After the gel dissolves, allow the solution to cool to room temperature, transfer the liquid to a column, and put it in the collection tube. Centrifuge for 60 seconds at 10,000 xg. Discard the flow-through liquid.
4. Add 650 μl WB, centrifuge for 60 seconds at 10,000 xg. Discard the flow-through liquid.
5. Centrifuge for 1-2 minutes at 10,000 xg to completely remove residual WB.
6. Place the centrifuge column in a clean centrifuge tube, open the cap and let it stand for 1 minute to allow the residual ethanol to evaporate completely. Then add 20 μl of deionized water (pre-warmed) to the center of the column and let it stand at room temperature for 1 minute.
7. Centrifuge for 60 seconds at 10,000 xg. Discard the column and store the result at -20℃.

### Gibson Assembly

**Goal:** To assemble the different DNA parts retrieved by PCR.

#### Materials:

* PCR tubes (200 μl)
* ClonExpress Ultra One Step Cloning Kit (2xClonExpress Mix, pCE-Zero vector linearized (50 ng/μl), 500bp control insert (20 ng/μl))
* ddH2O
* Thermocycler

#### Protocol:

1. Calculate the required amount of DNA for the recombination reaction according to the formula:
   * Single-fragment: Optimal cloning vector usage = [0.02 x number of base pairs of cloning vector] ng (0.03 pmol) Optimal insert fragment usage = [0.04 x number of base pairs of insert fragment] ng (0.06 pmol)
   * Multi-fragment: Optimal cloning vector usage = [0.02 x number of base pairs of cloning vector] ng (0.03 pmol) Optimal usage for each fragment = [0.02 x number of base pairs of each fragment] ng (0.03 pmol)
2. Prepare the following reaction system on ice:

   | Components | Recombination Reaction | Negative Control-1 | Negative Control-2 | Positive Control |
   | --- | --- | --- | --- | --- |
   | Linearized Vector | X μl | X μl | 0 μl | 1 μl |
   | n Insert Fragments (n≤5) | Y1+Y2...+Yn μl | 0 μl | Y1+Y2...+Yn μl | 1 μl |
   | 2x ClonExpress Mix | 5 μl | 0 μl | 0 μl | 5 μl |
   | ddH2O | to 10 μl | to 10 μl | to 10 μl | to 10 μl |
3. Gently mix well with a pipette (do not vortex mix), briefly centrifuge to collect the reaction liquid at the bottom of the tube.
4. Single-fragment recombination reaction, 50°C, 5 min; cool to 4°C or immediately place on ice.
5. Multi-fragment recombination reaction, 50°C, 15 min; cool to 4°C or immediately place on ice.

### RNA Extraction

#### Materials:

* StarSpin Plant RNA Kit (Buffer RPA, RNase-free DNase, Buffer DB, Buffer RW1, Buffer RW2, Buffer TB, Filtration Columns with Collection Tubes-RF, Spin Columns with Collection Tubes-RC, 1.5ml Centrifuge Tubes (DNase/RNase-free))

#### Protocol:

1. Take 500 μl of Buffer RPA (which has been supplemented with β-mercaptoethanol) and add it to a 1.5 ml RNase-free centrifuge tube. After grinding the tissue sample in liquid nitrogen, add the powdered sample (50-100 mg) to the 1.5ml centrifuge tube containing 500 μl of Buffer RPA. Vortex vigorously to mix well until there are no visible precipitates in the lysis solution.
2. Centrifuge at 12,000rpm for 2 minutes.
3. Place the filtration column into a collection tube, then transfer the supernatant collected from the previous centrifugation step into the Filtration Columns with Collection Tubes-RF (with the filtration column placed in the collection tube), centrifuge at 12,000 rpm for 2 minutes. Carefully aspirate the filtrate from the collection tube into a new 1.5 ml RNase-free centrifuge tube, avoiding contact with the cell debris precipitate in the collection tube.
4. Slowly add anhydrous ethanol equivalent to 0.4 times the volume of the filtrate. Mix well (a precipitate may form at this point), and transfer the resulting clear liquid and precipitate together into the 5pin Columns with Collection Tubes-RC (with the adsorption column placed in the collection tube), centrifuge at 12,000 rpm for 15 seconds, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.
5. Add 350 μl of Buffer RW1 to the adsorption column, centrifuge at 12,000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.
6. Prepare the RNase-free DNase working solution: take 10 μl of RNase-free DNase and add it to a new RNase-free centrifuge tube, add 70 μl of Buffer DB, and mix well to prepare an RNase-free DNase working solution with a final volume of 80 μl.
7. Apply 80 μl of the RNase-free DNase working solution to the center of the adsorption column and let it stand at room temperature for 15 minutes.
8. Add 350 μl of Buffer RW1 to the adsorption column, centrifuge at 12,000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.
9. Add 500 μl of Buffer RW2 (which has been supplemented with anhydrous ethanol) to the adsorption column, let it stand at room temperature for 2 minutes, and centrifuge at 12,000 rpm for 1 minute. Discard the waste liquid in the collection tube and place the adsorption column back into the collection tube.
10. Repeat step 9 once.
11. Place the adsorption column back into the collection tube and centrifuge at room temperature at 12,000 rpm for 3 minutes.
12. Place the adsorption column into a new 1.5ml centrifuge tube (DNase/RNase-free), add 50-100 μl of Buffer TB, let it stand at room temperature for 1-2 minutes, and centrifuge at 12,000 rpm for 1 minute to obtain the RNA solution. The RNA obtained should be used immediately or aliquoted and stored at -80℃ to avoid repeated freeze-thaw cycles.

### Reverse Transcription

**Goal:** Reverse extracted RNA into cDNA.

#### Materials:

* RNA template
* StarScript III All-in-one RT Mix with gDNA Remover Kit (StarScript III All-in-one RT Mix; 5×StarScript III All-in-one RT Buffer; Nuclease-free Water (DEPC-treated))
* 1.5ml Centrifuge Tubes (DNase/RNase-free)

#### Protocol:

| Substance | Volume |
| --- | --- |
| RNA template | ≤ 1 μg total RNA or ≤ 0.1 μg poly(A) mRNA |
| StarScript III All-in-one RT Mix | 1 μl |
| 5×StarScript III All-in-one RT Buffer | 4 μl |
| Nuclease-free Water (DEPC-treated) | up to 20 μl |

* React under the following conditions:

  | Temperature | Time |
  | --- | --- |
  | 37℃ | 2 min |
  | 50℃ | 15 min |
  | 80℃ | 2 min |
* The cDNA obtained after the reaction is completed is placed on ice for subsequent experiments or frozen storage.

### qPCR

**Goal:** Detecting the expression of target genes at the level of conversion rate.

#### Materials:

* DNA template
* Forward primer
* Reverse primer
* 2×RealStar Fast SYBR qPCR Mix (UNG, Low DNA) Kit (2×RealStar Fast SYBR qPCR Mix (UNG, Low DNA); High/Low ROX Reference Dye)
* Centrifuge Tubes
* Sterile Water

#### Protocol:

| Substance | Volume |
| --- | --- |
| DNA template | 1 μl |
| Forward primer | 0.5 μl |
| Reverse primer | 0.5 μl |
| 2×RealStar Fast SYBR qPCR Mix (UNG, Low DNA) | 10 μl |
| High/Low ROX Reference Dye | 0.4 μl |
| Sterile Water | up to 20 μl |

* React under the following conditions in real-time instrument:

  | Temperature | Time | Cycles |
  | --- | --- | --- |
  | 50℃ | 5 min |  |
  | 95℃ | 2 min |  |
  | 95℃ | 10 s | 40 |
  | 60℃ | 30 s |  |

# Microbiology Experiment

### 1. Transformation of chemically competent *E. coli*

#### Materials:

* LB medium
* LB agar plates with antibiotics (depends)
* Crushed ice
* Plasmid used to transform
* Chemically competent cell
* Centrifuge tube (1.5 mL)

#### Protocol:

1. Add 100 μl chemically competent cell and 10 μl of plasmid in the tube, mix gently and softly.
2. Incubate the tube for 30 minutes on ice.
3. Incubate for 90 seconds at 42°C, then immediately place on ice for 2 minutes.
4. Add 700-900 μl (depends) of LB liquid medium.
5. Incubate for 45 minutes under agitation at 200 rpm.
6. Centrifuge for 5 minutes at 6,000xg.
7. Remove 950 μl of supernatant and resuspend the bacteria with 50 μl of this supernatant.
8. Spread the solution on a LB agar plate containing the relevant antibiotic.
9. Incubate in incubator at 37°C overnight.
10. Check for colonies the next day.

### 2. General bacterial concentration testing

#### Materials:

* Nano spectrophotometer
* Test solution

#### Protocol:

1. Turn on the Nano spectrophotometer and select the measurement mode for bacterial suspension.
2. Calibrate the spectrophotometer with standard samples to ensure accuracy.
3. Add the bacterial suspension to a cuvette and place it in the spectrophotometer to measure the absorbance. Ensure cleanliness and optical path alignment.
4. Record the concentration of the bacterial suspension.

### 3. Bacterial plating

#### 3.1 Dilution coated plate method

#### Materials:

* Bacteria solution cultured for a period of time
* Solid medium
* Spreader

#### Protocol:

1. Add some bacteria solution onto the surface of the medium.
2. Spread from one side of the plate, draw a few circles, and then overlap the streaks. Use a sterilized spreader and ensure it’s not too hot.
3. Place the plates in the incubator with clean surfaces. Temperature varies by bacteria type.

#### 3.2 Plate streaking

#### Materials:

* Bacteria solution cultured for a period of time
* Solid medium
* Inoculating loop

#### Protocol:

1. Dip inoculating loop into some bacterial liquid.
2. Streak on one side of the plate, do a quarter turn, and then overlap the streaks.
3. Place the plates in the incubator with clean surfaces. Temperature varies by bacteria type.

### 4. Phenotype inducing

#### 4.1 Induced by IPTG

1. Culture the bacteria solution for 12 hours, ensuring OD600 is no less than 0.8.
2. Add 16 μl of 50 mg/ml IPTG.
3. After induction, culture at 37°C.
4. Sometimes, a non-induced control group is used.

#### 4.2 Microscopy

1. Observe smears using fluorescence and optical microscopes.

### Sudan Black Staining

1. Prepare 5 g/L Sudan Black staining solution.
2. Spread culture in thin film over slide.
3. Dry in air.
4. Pass slide through flame to fix.
5. Flood slide with stain; rinse and dry.
6. Place drop of oil on slide; examine with 100x objective.

### Triparental mating

### 6.1 Preparation: Bacterial Strain Activation and Liquid Culture

1. Activate the recipient strain *S. fredii* CCBAU45436 on TY+NA solid plates at 28°C for about 4-6 days until single colonies are visible. Inoculate the recipient strain to prepare the seed culture. (5-7 days before the experiment)
2. Activate the donor strain on LB+Gen solid plates; activate the helper strain on LB+Km at 37°C; inoculate the recipient strain in TY+NA tubes and culture at 28°C with shaking at 180 rpm. (1-2 days before the experiment)
3. Inoculate the donor strain in LB+Gen tubes and culture at 37°C with shaking at 200 rpm; inoculate the helper strain in LB+Km tubes and culture at 37°C with shaking at 200 rpm. (3-4 hours before the experiment)

### 6.2 First Experiment

1. Collect 1 mL of late-log phase recipient bacteria (rhizobia in TY medium, OD600 = 2~4), 0.5 mL each of mid-log phase donor and helper bacteria (E. coli in LB medium, OD600 = 0.5-1.0) in a 1.5 mL centrifuge tube, and centrifuge at 12,000 rpm or 13,000 x g at room temperature for 1 minute.
2. Resuspend and mix all bacteria with 1 mL of sterile TY liquid medium or sterile saline (vortex to mix well), and centrifuge at 12,000 rpm at room temperature for 1 minute. Discard the supernatant to remove residual antibiotics from the medium.
3. Repeat last step to wash the bacteria once more.
4. Resuspend and combine all bacteria obtained from the centrifugation with 1 mL of saline (or TY liquid medium), and centrifuge at 12,000 rpm at room temperature for 1 minute.
5. Retain about 30-50 μL of the supernatant, mix and vortex the bacterial pellet well, spot it onto the center of a TY agar plate without antibiotics; after the bacterial spot dries and stops spreading, invert and incubate at 28°C for 20-40 hours.

### 6.3 Second Experiment

1. Observe and record the growth of bacteria on TY+NA+Gen plates (complete photography and counting after subsequent sterile operations, select plates with a bacterial count between 30-300 for counting).
2. Use sterilized white or yellow pipette tips to pick a small amount of single-crossover bacterial cells from the TY+NA+Gen plates (select from plates with lower colony density) and inoculate into TY liquid medium (without antibiotics or with NA only), culture at 28°C with shaking at 180 rpm for 24-30 hours.

### 6.4 Third Experiment

1. Observe and record the growth of double-crossover colonies on TY+sucrose+NA plates (photograph and count after completing subsequent sterile operations, select plates with a colony count between 30-300 for enumeration).
2. Use sterilized toothpicks (white/yellow pipette tips) to randomly select 3 large colonies (wild type) and 3 small colonies (mutants), and transfer them to clean TY+NA+5% sucrose solid medium plates for incubation at 28°C for 2-3 days.

### Touchdown PCR

#### Materials:

* PCR tubes (200 μl)
* 2 x Taq Mix
* MucR1-WL (10 μM)
* Template
* ddH2O
* Thermocycler

#### Protocol:

1. Add in a PCR tube:

   | Substance | Volume |
   | --- | --- |
   | 2 x Taq Mix | 12 μl |
   | MucR1-WL (10 μM) | 1 μl |
   | MucR1-WL (10 μM) | 1 μl |
   | ddH2O | 11 μl |
2. Use a white pipette tip to take a small amount of bacterial cells (0.1 μL) as a DNA template.
3. Except for the template, mix in advance in an n-fold quantity and then aliquot.
4. Place the tube in the thermocycler and start corresponding program.

### Agarose Gel Electrophoresis Analysis

1. Gel Preparation (1.5%)
2. Electrophoresis: 3-5 μl of sample, 5 μl of marker, constant voltage of 150V for 30-60 minutes.

# Plant Experiment

### 1. Prepare Low-Nitrogen Nutrient Solution

Prepare the low-nitrogen nutrient solution according to the following recipe for every 100 seedlings (500 mL needed).

| Substance | Volume |
| --- | --- |
| Ca(NO3)2·4H2O | 0.6 g/L |
| KCl | 1.5 g/L |
| MgSO4·7H2O | 2.46 g/L |
| K2HPO4·3H2O | 3.56 g/L |
| Microelement (1x) | 20 ml/L |
| CaSO4·2H2O | 9.2 g/L |
| Ferric citrate·5H2O | 2.05 g/L |

### 2. Container-related Preparation

1. Thread the cup with a gauze strip.
2. Divide the water into portions and sterilize at high temperature for 30 minutes.
3. Mix with vermiculite (1 large bag of vermiculite is needed for every 100 seedlings) and sterilize.

### 3. Seed-related Preparation and Inoculate the Seedlings

1. Select seeds (For planting x number of seedlings, it is best to select 3x number of seeds).
2. Disinfect the seeds (with chlorine gas disinfection method).
3. Prepare 0.8% water agar, sterilize, pour plates, and germinate seeds.
4. Plant the seedlings. The device is shown in the figure below.
5. Begin shaking the culture 24 hours before inoculation, and the OD value should be around 2. Collect the bacteria. Inoculate the bean seedlings.
6. Regularly observe the growth, supplement water and nutrients as needed, adjust lighting, and harvest.

# Others

### 1. Fluorospectrophotometer

#### Materials:

* F93 Fluorospectrophotometer (Lengguang Tech)
* Test samples
* Reference solution

#### Protocol:

1. Preheat the main unit for 30 minutes.
2. Adjust to the required wavelength.
3. Select the fluorescence value measurement function.
4. Set sensitivity and zero adjustment.
5. Open the sample chamber cover, remove cuvette one, place cuvette two into the cuvette holder, and close the sample chamber cover.
6. Read and record the measurement.
7. Proceed to measure other samples, or turn off the main power switch of the instrument.

### 2. Mass Spectrum

#### Materials:

* Liquid nitrogen
* Test samples
* 50% methanol
* Ultrasonic cell disruptor
* Centrifuge
* Filtration apparatus

#### Protocol:

1. Grind into a powder using liquid nitrogen.
2. Weigh out an equal amount of the sample, 100mg, add 1ml of 50% methanol, and sonicate for 30 minutes.
3. Centrifuge at 14,000 rpm for 10 minutes.
4. Take the supernatant and filter it to obtain the filtrate, then perform a concentration gradient dilution with 50% methanol.
5. Carry out direct mass spectrometry analysis. (Completed by the Mass Spectrometry Platform of the College of Biological Sciences, China Agricultural University.)

### 3. Transmission Electron Microscope

#### Materials:

* Liquid nitrogen
* Test samples
* 50% methanol
* Ultrasonic cell disruptor
* Centrifuge
* Filtration apparatus

#### Protocol:

1. *S. fredii* are cultured in TY broth without antibiotics until they reach the stationary phase.
2. Centrifuge at 8,000xg for 5 minutes to collect 1 ml of bacterial cells.
3. Discard the supernatant.
4. Resuspend the cells in 2.5% glutaraldehyde for fixation.
5. Proceed with subsequent transmission electron microscopy operations. (Completed by the Mass Spectrometry Platform of the College of Biological Sciences, China Agricultural University.)

### 4. Thin-Layer Chromatography (TLC)

#### Materials:

* 0.1 M PBS
* Methanol
* Chloroform
* 0.9% Sodium chloride solution
* Nitrogen
* n-Hexane
* Methyl tert-butyl ether
* Acetic acid
* 8% Phosphoric acid
* 10% CuSO4 · 5H20

#### Protocol:

1. Collect bacteria
   1. Collect 10 mL of bacterial solution into a pre-weighed centrifuge tube.
   2. Centrifuge at 8000 rpm for 1 minute to collect the bacterial cells.
   3. Wash twice with 0.1 M PBS.
   4. Freeze in liquid nitrogen and calculate the dry weight of the bacterial cells after vacuum freeze-drying.
2. Extract oil
   1. Add 1ml of methanol to 25 mg of bacterial cells, and react for 5 minutes.
   2. Add 3ml of chloroform and 0.5 ml of 0.9% sodium chloride solution, shake and mix well.
   3. Centrifuge at 3000 rpm for 6 minutes.
   4. Transfer the lower organic phase to a pre-weighed glass test tube.
   5. Dry the organic phase with nitrogen, and weigh to calculate the amount of extracted oil.
3. Chromatography
   1. Redissolve the grease with chloroform in the ratio of 1:20.
   2. Take 10 μl of spot sample to the Silica gel 60 F254 plate.
   3. After the spot sample is completed, prepare the developing agent: n-hexane: methyl tert-butyl ether: acetic acid=80:20:2 (v/v/v, currently prepared and used).
   4. Spread for 20 minutes, take out the plate 15 cm from the upper edge, and blow dry.
   5. Show the color in chromogenic solution (8% phosphoric acid and 10% CuSO4·5H20) for 30 seconds, take out and blow dry.
   6. Bake at 180 °C for 3 minutes.
   7. Record with the gel imager.

{% endblock %}
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{% endblock %}

{% extends "layout.html" %}
{% block title %}Team{% endblock %}
{% block lead %}On this page you can introduce your team members, instructors, and advisors.{% endblock %}
{% block tstyle %}

{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5300/source/results/top.png)

[Overview](#Overview)

[PHB Deletion](#PHB)

[Regulation module](#Regulation)

[Synthesis module](#Synthesis)

[Suicide circuit](#Suicide)

# Overview

CAU-China aimed to utilize nodule's own anaerobic environment to create an anaerobic biofactory, providing new ideas for solving the problem faced by anaerobic or hypoxic production. The purpose of this page is to present the results of four modules to provide experimental evidence for the validation of the ideas.

# PHB Deletion

In order to provide sufficient raw material for the synthesis of the target products, we attempted to delete **phaC2**, a gene encoding an important enzyme in the PHB synthesis pathway, thereby increasing the amount of raw material for the synthesis of the target products.

### Plasmid construction and validation

Firstly, the upstream and downstream about 500 bp respectively of **phaC2** gene were used as UP arm[(BBa\_K5300029)](https://parts.igem.org/Part%3ABBa_K5300029) and Down arm[(BBa\_K5300030)](https://parts.igem.org/Part%3ABBa_K5300030) to construct ‘Up-Arm **phaC2** + Down-Arm **phaC2*’*[(BBa\_K5300040)](https://parts.igem.org/Part%3ABBa_K5300040) fragment, and Gibson was used to introduce the composite fragment into vector pJQ200SK linearized by SmaI monoenzyme cleavage, and the recombinant plasmid was introduced into DH5α using the method of transformation, and coated onto gentamicin-resistant LB solid medium, incubated at 37°C overnight and then subjected to colony PCR using the universal primer M13F/R (Figure 1-1-1).

![](https://static.igem.wiki/teams/5300/source/experiment/1-1-1.png)

**Fig.1-1-1 The results of colony PCR**. Positive colonies were expected at around 1.2kb, which all six picked colonies from the Gibson assembly turned out to be.

Colony PCR proved that the recombinant plasmid was successfully introduced, and the correct colonies were inoculated into gentamicin-resistant LB liquid medium under shaking condition overnight, and the plasmids were extracted and sent for sequencing on the second day, whose results proved that the plasmids were constructed correctly and successfully.

### Triparental mating and validation

The verified correct colonies were inoculated into gentamicin-resistant LB liquid medium under shaking conditions and cultured, triparental mating was performed using Helper, *Sinorhizobium fredii* CCBAU45436, and two screenings were performed. The single colonies obtained from the second screening were subjected to colony PCR (Figure 1-2-1).

![](https://static.igem.wiki/teams/5300/source/experiment/1-2-1.png)

**Fig. 1-2-1 The results of colony PCR.**. Positive colonies were expected at around 1.2kb, which two picked colonies in the lane 4-5 turned out to be. Two colonies in the lane 2-3 are wild type of bacteria.

Colony PCR demonstrated that the UA and DA portions of the plasmid underwent homologous recombination with *Sinorhizobium fredii* CCBAU45436, and the **phaC2** gene was successfully deleted. Notably, the *∆*phaC2** mutants showed different phenotype from the wild type. The colonies were seen to be distinctly yellow when the bacteria were re-picked and streaked on TY solid medium (Figure 1-2-2).

![](https://static.igem.wiki/teams/5300/source/experiment/1-2-2.png)

**Fig. 1-2-2 The phenotypes of two types of bacteria.** . (a) *Sinorhizobium fredii* CCBAU45436. (b) *∆*phaC2**

### Plant system validation

The deletion of **phaC2** resulted in obvious phenotype changes in *Sinorhizobium fredii* CCBAU45436, and we subsequently inoculated the mutant on Jidou 17 to test whether its original nitrogen-fixing function had been altered. After 15 days of incubation, photos were taken to record the above-ground phenotype, the below-ground phenotype (Figure 1-3-1), and the rhizomatous section (Figure 1-3-2).

![](https://static.igem.wiki/teams/5300/source/experiment/1-3-1.png)

**Fig. 1-3-1 Plant phenotype after 15 days of growth.**(a1-2) Not inoculated with rhizobium. (b1-2) Inoculated with *Sinorhizobium fredii* CCBAU45436. (c1-2) Inoculated with *∆*phaC2**

![](https://static.igem.wiki/teams/5300/source/experiment/1-3-2.png)

**Fig. 1-3-2 Section of nodules after 15 days of growth.**(a) Inoculated with *Sinorhizobium fredii* CCBAU45436. (b) Inoculated with *∆*phaC2**

There were no significant differences between wild-type and mutant-inoculated plants on phenotype; while mutant strains showed darker color for rhizomatous sections. Their chlorophyll content and dry weight of above-ground parts were examined (Figure 1-3-3).

![](https://static.igem.wiki/teams/5300/source/experiment/1-3-3.png)

**Fig. 1-3-3 Plant phenotypes for nodulation experiments after 15 days of growth.**(a) Chlorophyll content of plants. (b) Dry weight of the above-ground part. Student’s t-test, ns: no significant difference; \*, p-value < 0.05; \*\*, p-value&lt0.01 \*\*\*, p-value < 0.001.

Analyzing the data of chlorophyll content and dry weight of above-ground parts after 15-day growth, none of the three groups, blank control, wild type and mutant, showed significant differences, indicating that it was not possible to prove whether the differences occurred or not under 15 days of growth. Therefore, a second round of plant experiment was carried out and verified after 21 days of culture after inoculation (Figure 1-3-4).

![](https://static.igem.wiki/teams/5300/source/experiment/1-3-4.png)

**Fig. 1-3-4 Plant phenotype for nodulation experiments after 21 days of growth.** (a) Chlorophyll content of plants. (b) Dry weight of the above-ground part. Student’s t-test, ns: no significant difference; \*, p-value < 0.05; \*\*, p-value&lt0.01 \*\*\*, p-value < 0.001.

Chlorophyll content and above-ground part dry weight data from the second round of plant experiments demonstrated that the *∆*phaC2** mutant does not affect the growth of the above-ground part of Jidou 17.

### Electron microscope scan

To further observe the morphology of the mutant strain, we chose the bacterial solution of the platform stage for transmission electron microscopy observation and counted the number of its PHB particles, whose results are shown in Figure 1-4-1 and Figure 1-4-2.

![](https://static.igem.wiki/teams/5300/source/experiment/1-4-1.jpg)

**Fig. 1-4-1 The results of TEM.** (a-b) *Sinorhizobium fredii* CCBAU45436. (c-d) *∆*phaC2**

![](https://static.igem.wiki/teams/5300/source/experiment/1-4-2.png)

**Fig. 1-4-2 The results of data statistics.**

The number of PHB particles in the wild-type strain and the mutant strain showed a significant difference, further validating the success of the **phaC2** deletion module.

### Total Lipid TLC Analysis

In order to analyze the changes in fatty acid content in *∆*phaC2** strains, we used thin-layer chromatography to analyze the amount of triacylglycerol (TAG) in *∆*phaC2** strains as a proxy for fatty acid content (Figure 1-5-1).

![](https://static.igem.wiki/teams/5300/source/experiment/1-5-1.png)

**Fig. 1-5-1 The results of TLC.**

*∆*phaC2** strains were able to show a significant increase in the content of TAG compared to the wild type. We believed that this is due to the altered fatty acid content. This is in line with our design. The deletion of *phaC2* gene leads to an increase in the substrates for fatty acid synthesis, which in turn leads to an increase in fatty acid content.

# Regulation module

In order to ensure that the synthesis of the target product is accomplished without affecting the nitrogen-fixing function of the rhizobium itself, we designed the regulation module to enable the induction of the synthesis of the target product under dual conditions so as to utilize the rhizobial system with the highest efficiency.

### Promoter validation

First, we validated the *glnK* promoter [(BBa\_K5300017)](https://parts.igem.org/Part%3ABBa_K5300017) alone and explored its expression under different nitrogen concentrations. We constructed a “promoter + fluorescent protein” system in pBBR1MCS-2 (Figure 2-1-1) to reflect the ability of the promoter based on the expression of the fluorescent protein. Since *glnK* is regulated by nitrogen in both rhizobium and *E. coli* and the growth cycle of rhizobium is longer, we chose to validate the system in *E. coli*. The successfully constructed plasmid was introduced into *E. coli* (DH5α) and colony PCR was performed for verification (Figure 2-1-2).

![](https://static.igem.wiki/teams/5300/source/experiment/2-1-1.png)

**Fig. 2-1-1 The model of promoter validation. [(BBa\_K5300045)](https://parts.igem.org/Part%3ABBa_K5300045)**

![](https://static.igem.wiki/teams/5300/source/experiment/2-1-2.png)

**Fig. 2-1-2 The results of colony PCR.**Positive colonies were expected at around 1.5kb, which all ten picked colonies from the Gibson assembly turned out to be.

Subsequently, we explored the induction conditions by using M9 medium without nitrogen source as culture conditions and adding different concentrations of ammonium chloride as an additional nitrogen source. The OD600 value of the bacterial solution was determined after overnight shock incubation, and the fluorescence intensity of the bacterial solution was measured using a fluorescence spectrophotometer (Figure 2-1-3).

![](https://static.igem.wiki/teams/5300/source/experiment/2-1-3.png)

**Fig. 2-1-3 Ratio of relative fluorescence intensity to OD600 value of bacterial solution.** (a) Column diagram. (b)Broken line graph. Student’s t-test, ns: no significant difference; \*, p-value < 0.05; \*\*, p-value&lt0.01 \*\*\*, p-value < 0.001.

Obviously, there was a significant difference between the fluorescence expression intensity under nitrogen-free conditions and that in the presence of a rich nitrogen source, successfully verifying that the *glnK* promoter was repressed by high nitrogen conditions.

In order to verify the function of *nifH* [(BBa\_K5300016)](https://parts.igem.org/Part%3ABBa_K5300016) promoter, we constructed a composite circuit of *nifH* promoter and *gfp* [(BBa\_K5300015)](https://parts.igem.org/Part%3ABBa_K5300015), which was seamlessly cloned into pBBR1MCS-2 [(BBa\_K5300023)](https://parts.igem.org/Part%3ABBa_K5300023) along with “*glnK* promoter+sgRNA-GFP [(BBa\_K5300025)](https://parts.igem.org/Part%3ABBa_K5300025) for subsequent experiments. (Figure 2-1-4).

![](https://static.igem.wiki/teams/5300/source/experiment/2-1-4.png)

**Fig. 2-1-4 The model of promoter validation. [(BBa\_K5300043)](https://parts.igem.org/Part%3ABBa_K5300043)**

Since *Cas12k* [(BBa\_K5300021)](https://parts.igem.org/Part%3ABBa_K5300021) was not expressed and the *glnK* promoter was repressed by high-nitrogen conditions, we cultured rhizobia with this plasmid in high-nitrogen (1 g/L NH4Cl) M9 medium. Under these conditions it was thought that the *glnK* promoter would be unable to express subsequent genes and that *gfp* expression would not be repressed. The fluorescence intensity was detected after 24 h of incubation under both low and high oxygen conditions, respectively (Figure 2-1-5).

![](https://static.igem.wiki/teams/5300/source/experiment/2-1-5.png)

**Fig. 2-1-5 Ratio of relative fluorescence intensity to OD600 value of bacterial solution.** Student’s t-test, ns: no significant difference; \*, p-value < 0.05; \*\*, p-value < 0.01; \*\*\*, p-value < 0.001.

Apparently, the expression intensity of *nifH* promoter was up-regulated under low-oxygen conditions; while under high-oxygen conditions, the expression intensity of *nifH* promoter was down-regulated. It was successfully verified that the *nifH* promoter was repressed by hyperoxic conditions.

### Plasmid construction and validation

We first amplified the *glnK* promoter and *nifH* promoter from the genome of *Sinorhizobium fredii* CCBAU45436, amplified *gfp* and designed sgRNAs from pRJPaph-bjGFP plasmid, and ligated them using overlap PCR. To verify the effect of the regulation module alone, we connected it with the *nuoA* [(BBa\_K5300013)](https://parts.igem.org/Part%3ABBa_K5300013) promoter-*Cas12k* [(BBa\_K5300021)](https://parts.igem.org/Part%3ABBa_K5300021) and the linearized pBBR1MCS-2 (Figure 2-2-1). The recombinant plasmid was transformed into *E. coli* (DH5α).

![](https://static.igem.wiki/teams/5300/source/experiment/2-2-1.png)

**Fig. 2-2-1 The model of validation of regulation circuit. [(BBa\_K5300044)](https://parts.igem.org/Part%3ABBa_K5300044)**

### Triparental mating and validation

The verified correct colonies were inoculated into kanamycin-resistant LB liquid medium under shaking condition. Then we conducted triparental mating using the correct colony we obtained above, Helper and *Sinorhizobium fredii* CCBAU45436. Then colony PCR was performed on colonies grown on TY/NA/Kan solid medium incubated at 28°C for 24 h using the universal primer M13F/R.

### Fluorescence intensity verification

We utilized its fluorescence expression intensity to verify whether the regulation module functioned properly. We set up four sets of experiment with high nitrogen and high oxygen, low nitrogen and high oxygen, high nitrogen and low oxygen, as well as low nitrogen and low oxygen for verification. Subsequently, we used M9 medium without nitrogen source,then added different concentrations of ammonium chloride to form nitrogen concentration gradient. We added petroleum jelly after boiling the medium to isolate medium against atmosphere, creating anaerobic conditions. The OD600 value of the bacterial solution was determined after a period of shaking and incubation, and the fluorescence intensity of the bacterial solution was determined using a fluorescence spectrophotometer (Figure 2-4-1 and Figure 2-4-2).

![](https://static.igem.wiki/teams/5300/source/experiment/2-4-1.png)

**Fig. 2-4-1 Ratio of relative fluorescence intensity to OD600 value of bacterial solution.** (a) Different oxygen conditions at low nitrogen (0 g/L NH4Cl). (b) Different oxygen conditions at high nitrogen (0.02 g/L NH4Cl). (c) Different conditions of nitrogen concentration during hypoxia. (d) Different conditions of nitrogen concentration during hyperoxia. Student’s t-test, ns: no significant difference; \*, p-value < 0.05; \*\*, p-value&lt0.01 \*\*\*, p-value < 0.001.

![](https://static.igem.wiki/teams/5300/source/experiment/2-4-2.png)

**Fig. 2-4-2 Ratio of relative fluorescence intensity to OD600 value of bacterial solution.** Student’s t-test, ns: no significant difference; \*, p-value < 0.05; \*\*, p-value&lt0.01 \*\*\*, p-value < 0.001.

We were able to clearly see that high oxygen conditions always inhibit the expression of fluorescent proteins, whether under high or low nitrogen conditions. Low nitrogen conditions also inhibit the expression of fluorescent proteins, whether under high or low oxygen conditions. This is consistent with our assumptions. Under high oxygen conditions, the *nifH* promoter is repressed, which reduces the expression of downstream genes, thereby the fluorescence intensity is reduced. Under low nitrogen conditions, the *glnK* promoter is promoted, and the expression of sgRNA is up-regulated, directing the *Cas12k* protein to repress the expression of fluorescent proteins. However, both low-nitrogen and high-oxygen conditions induced high expression, which is hypothesized to be the result of uninhibited constitutive expression of the promoter, as well as the possibility of leaky expression.

# Synthesis module

In order to verify the validity of the “nodule factory”, we chose to utilize it for the production of dipeptidyl aldehyde and PUFA (namely polyunsaturated fatty acids).

### Dipeptide aldehyde

#### Plant system validation

In order to verify that our idea works properly, we first conducted the synthesis of dipeptide aldehydes. We constructed a composite circuit of *nifH* promoter and dipeptide aldehyde synthesis gene cluster (Figure 3-1-1). To verify whether the plasmid would stress the growth of nodules, we constructed a composite circuit of the *nifH* promoter and half of the sequence of the dipeptidyl aldehyde synthesis gene cluster (Fig. 3-1-2), which encodes a product that cannot synthesize complete dipeptidyl aldehyde.

![](https://static.igem.wiki/teams/5300/source/experiment/3-1-1.png)

**Fig. 3-1-1 The model 1 of validation of dipeptidyl aldehyde synthesis.**

[(BBa\_K5300039)](https://parts.igem.org/Part%3ABBa_K5300039)

![](https://static.igem.wiki/teams/5300/source/experiment/3-1-2.png)

**Fig. 3-1-2 The model 2 of validation of dipeptidyl aldehyde synthesis.**

The plasmid for dipeptide aldehyde synthesis in *E. coli* was transferred into *Sinorhizobium fredii* CCBAU45436 using triparental mating. The verified correct strain was inoculated into Jidou 17 and harvested after 21 days of growth, and photographs were taken to record the above-ground phenotype, below-ground phenotype (Fig. 3-1-3), and nodules section (Fig. 3-1-4).

![](https://static.igem.wiki/teams/5300/source/experiment/3-1-3.png)

**Fig. 3-1-3 Plant phenotype after 21 days of growth.** (a1-2) Not inoculated with rhizobium. (b1-2) Inoculated with *Sinorhizobium fredii* CCBAU45436. (c1-2) Inoculated with *Sinorhizobium fredii* CCBAU45436-*PnifH+bgc33*. (d1-2) Inoculated with *Sinorhizobium fredii* CCBAU45436-*PnifH+bgc33+sfp*

![](https://static.igem.wiki/teams/5300/source/experiment/3-1-4.png)

**Fig. 3-1-4 Section of nodules after 21 days of growth.**  (a) Inoculated with *Sinorhizobium fredii* CCBAU45436. (b) Inoculated with *Sinorhizobium fredii* CCBAU45436-*PnifH+bgc33*. (c) Inoculated with *Sinorhizobium fredii* CCBAU45436-*PnifH+bgc33+sfp*.

For phenotypes and nodule sections, there were no significant differences between plants inoculated with wild type and strains containing dipeptide aldehyde synthesis plasmid. Their chlorophyll content in leaves and dry weight of above-ground parts were examined (Figure 3-1-5).

![](https://static.igem.wiki/teams/5300/source/experiment/3-1-5.png)

**Fig. 3-1-5 Plant phenotypes for nodulation experiments after 21 days of growth.**  (a) Chlorophyll content of plants. (b) Dry weight of the above-ground part. Student’s t-test, ns: no significant difference; \*, p-value < 0.05; \*\*, p-value&lt0.01 \*\*\*, p-value < 0.001.

For chlorophyll content of plants after 21 days of growth, there was a significant difference between plants inoculated with rhizobium and the blank control. There was no significant difference between the different strains. For dry weight of above-ground portions, inoculation with recombinant-plasmid-containing bacteria led to a decrease in dry weight, whereas whether or not dipeptidyl aldehydes were synthesized did not affect dry weight of above-ground portions.

#### Real-time fluorescence quantitative PCR

We used RT-qPCR to quantify gene transcription levels. We extracted RNA from +*bgc33-sfp* strain and wild-type strain grown for 21 days respectively and conducted reverse transcription. qPCR was performed using cDNA as a template. 16S rRNA was used as an internal reference to compare the expression of the target gene in the +*bgc33-sfp* [(BBa\_K5300032)](https://parts.igem.org/Part%3ABBa_K5300032) strain with the endogenous *nifH* gene. We designed three pairs of primers (bgc33-a, bgc33-b, and bgc33-c) for *bgc33* [(BBa\_K5300031)](https://parts.igem.org/Part%3ABBa_K5300031) gene to characterize the transcript levels of different regions of the gene in terms of the transcript levels of three fragments, and performed three biological replicates (Figure 3-2-1).

![](https://static.igem.wiki/teams/5300/source/experiment/3-2-1.png)

**Fig. 3-2-1 The results of real-time fluorescence quantitative PCR.** (a) Chlorophyll content of plants. (b) Dry weight of the above-ground part. Student’s t-test, ns: no significant difference; \*, p-value < 0.05; \*\*, p-value&lt0.01 \*\*\*, p-value < 0.001.

Obviously, we found that the expression of *bgc33* gene decreased following the increase of its distance from the promoter. The transcript levels of the first two segments of *bgc33* gene were up-regulated compared to the expression of the endogenous *nifH* gene, while the expression of the third segment was down-regulated. We hypothesized that the decreased transcript levels of the second half of the gene may be due to the fact that the gene is long and the mRNA is susceptible to degradation or forms secondary structure during transcription, which leads to post-transcriptional instability in complete translation of the gene.

#### Mass Spectrometry Results

To verify whether the gene cluster was phenotypically successful in synthesizing dipeptide aldehydes, we used direct mass spectrometry to analyze the mass-to-charge ratio of dipeptide aldehydes (Figure 3-3-1).

![](https://static.igem.wiki/teams/5300/source/experiment/3-3-1.png)

**Fig. 3-3-1 Structure and mass-charge ratio of dipeptidyl aldehyde.**  (a) The structure of dipeptidyl aldehyde. Dipeptide aldehyde, Met-Phe-H, exists in equilibrium with the cyclic imine. In the presence of oxygen, this dihydropyrazinone oxidizes irreversibly to the pyrazinone. (b) The mass-charge ratio of dipeptidyl aldehyde. (c) The results of mass spectrometry

We performed direct mass spectrometry on the wild-type strain and the +*bgc33-sfp* strain and analyzed the +*bgc33-sfp* strain according to a gradient of 100-fold dilution, 10-fold dilution of the solution and the stock solution. We did not find new peaks in the diluted liquid, but surprisingly, when we used the stock solution, we detected new peaks not found in the wild type. Although the amount was extremely low, we finally found the new product. Based on the results of real-time fluorescence quantitative PCR, we believed that the amount of products is greatly reduced under the influence of low transcription levels, and therefore we could only detect peaks with a very small area.

### PUFA

#### Plasmid construction and validation

We first seamlessly cloned the regulation module and each two fragments of PUFA1-5 with the PCR linearized pUC19 vector in order to amplify three large fragments (i.e., regulation-PUFA1, PUFA2-3, and PUFA4-5) using the recombinant plasmid as a template. The three large fragments were seamlessly cloned with the PCR linearized pBBR1MCS-2 cloning, and the recombinant plasmids were transformed into *E. coli* (DH5α) (Figure 3-4-1).

![](https://static.igem.wiki/teams/5300/source/experiment/3-4-1.png)

**Fig. 3-4-1 The model of synthesis module. [(BBa\_K5300042)](https://parts.igem.org/Part%3ABBa_K5300042)**

The sequencing results of this plasmid were disappointing, as it showed several point mutations and even nonsense mutations, making our experiment impossible. We were unable to repeat the experiment again due to time constraints. And in order to validate our synthesis module, we therefore designed a synthesis circuit that utilizes a constitutive promoter for expression. We repaired the point mutation in the pfa biosynthetic gene cluster and seamlessly cloned and ligated it to the nuoA promoter to pBBR1MCS-2. (Figure 3-4-2)

![](https://static.igem.wiki/teams/5300/source/experiment/3-4-2.png)

**Fig. 3-4-2 The modified model of synthesis module. [(BBa\_K5300037)](https://parts.igem.org/Part%3ABBa_K5300037)**

#### Triparental mating and validation

The verified correct colonies were inoculated into gentamicin-resistant LB liquid medium under shaking conditions and cultured, triparental mating was performed using Helper, *Sinorhizobium fredii* CCBAU45436, and two screenings were performed. The single colonies obtained from the second screening were subjected to colony PCR utilizing the universal primer M13F/R.

#### RT-qPCR results

We used RT-qPCR to quantify gene transcription levels. We extracted RNA from the bacterial solution of the +*pfa* strain and the wild-type strain, respectively and conducted reverse transcription. qPCR was performed using cDNA as a template. 16S rRNA was used as an internal reference to compare the expression of the target genes in the +*pfa* strain relative to the endogenous *nuoA* gene. We designed three pairs of primers (pfa1, pfa2, pfa3) of the three core enzyme genes of the *pfa* gene to characterize the amount of transcription in different segments of the gene in terms of the transcript levels of the three fragments and performed three biological replicates (Figure 3-6-1).

![](https://static.igem.wiki/teams/5300/source/experiment/3-6-1.png)

**Fig. 3-6-1 The results of real-time fluorescence quantitative PCR.**Student’s t-test, ns: no significant difference; \*, p-value < 0.05; \*\*, p-value&lt0.01 \*\*\*, p-value < 0.001.

Obviously, we found that the expression of the *pfa* gene decreased following the increase of its distance from the promoter. The transcript levels of all three segments of *pfa* gene were up-regulated compared to the expression of the endogenous *nuoA* gene, but the overall trend was a gradual decrease. We hypothesized that the decrease in transcript levels in the second half of the gene may be due to the fact that the gene is long and the mRNA is prone to degradation or forms secondary structure during transcription, which leads to post-transcriptional instability in complete translation.

#### Total lipid TLC analysis

In order to pre-analyze the changes in fatty acid content in the +*pfa* strain, we analyzed the amount of triacylglycerol (TAG) in the +*pfa* strain using thin-layer chromatography to represent the fatty acid content (Figure 3-7-1).

![](https://static.igem.wiki/teams/5300/source/experiment/3-7-1.png)

**Fig. 3-7-1 The results of TLC.**

#### Gas Chromatography Results

In order to directly verify that the gene cluster was actually successfully expressed and synthesized the target product, we used gas chromatography to isolate, purify and characterize the fatty acid content above ten carbons. We sent the wild-type strain and the +*pfa* strain for gas chromatographic identification (Figure 3-8-1).

![](https://static.igem.wiki/teams/5300/source/experiment/3-8-1.png)

**Fig. 3-8-1 The results of gas chromatography.**

It’s a pity that we did not identify the presence of DHA and EPA. But with the bad news came good news. We identified a fatty acid in the +*pfa* strain that was not present in the wild-type strain - a long-chain saturated fatty acid with fourteen carbons. And the content of this fatty acid varied dramatically, from nonexistent to 5% of the total. We read the references and found that the expression of gene cluster is very complex. *pfa* biosynthetic gene clusters are capable of expressing several enzymes, each plays a different role (Figure 3-8-2).

![](https://static.igem.wiki/teams/5300/source/experiment/3-8-2.jpg)

**Fig. 3-8-2 Anaerobic biosynthesis of polyunsaturated fatty acids (PUFAs) by iterative type I fatty acid synthase (FAS)/polyketide synthase (PKS)-like PUFA synthases encoded by a *pfa* biosynthetic gene cluster.** The primer molecule (acetyl-CoA) is extended by several rounds of decarboxylative Claisen condensation reactions, resulting in the elongation of the fatty acyl chain by two carbons (derived from malonyl-CoA) per cycle. Following each round of elongation, the β-keto group is either fully reduced or only reduced to the *trans* double bond which is then isomerized. Finally, an acyl chain with methylene-interrupted cis double bonds is synthesized. AT=acyltransferase, ACP=acyl carrier protein, KS=ketosynthase, KR=ketoreductase, DH=dehydratase/isomerase, ER=enoylreductase. (From Katja Gemperlein, 2016)

Since new fatty acids that never appeared in the wild type appeared in our +*pfa* strain, it indicates that the expression of the gene cluster was successful to some extent. Based on the results of real-time fluorescence quantitative PCR, we analyzed that the failure to produce the target product was due to the greatly reduced transcription of the second half of the cluster. In Figure 3-8-2, we found that the oxidase was encoded only by *pfa3 [(BBa\_K5300009)](https://parts.igem.org/Part%3ABBa_K5300009)*, which is capable of forming unsaturated bonds. Also, the gas chromatography we used for time reasons was food grade and less accurate. Therefore there is a high probability that *pfa3* failed to express the oxidase in large quantities thus DHA and EPA were not detected with the low precision of the assay.

To verify our speculation, we organized the data and reanalyzed it. *pfa1* [(BBa\_K5300004)](https://parts.igem.org/Part%3ABBa_K5300004) expresses the product of ER, the reductase, which is able to change unsaturated bonds to saturated bonds. We therefore counted the proportion of unsaturated fatty acids among the eighteen-carbon fatty acids and the changes in the content of different types of fatty acids (Fig. 3-8-3).

![](https://static.igem.wiki/teams/5300/source/experiment/3-8-3.png)

**Fig. 3-8-3 The results of data statistics.**(a) Changes in the content of different types of fatty acids. (b) Ratio of C18 unsaturated fatty acid contents to C18 fatty acid contents.

Obviously, we found that the content of unsaturated fatty acids in the +*pfa* strain decreased significantly compared to the wild type strain, which is in line with our speculation. Therefore, it can be proved to a certain extent that our synthesis module is successful and the related genes are able to be expressed normally. And in order to obtain the final target product as well as to increase the yield, it is necessary to optimize the subsequent experiments.

# Suicide circuit

### Plasmid construction and validation

We first amplified the *glnK* promoter, *nifH* promoter, *vapC* [(BBa\_K5300019)](https://parts.igem.org/Part%3ABBa_K5300019) and *vapB* [(BBa\_K5300018)](https://parts.igem.org/Part%3ABBa_K5300018) from the genome, amplified mCherry [(BBa\_K5300014)](https://parts.igem.org/Part%3ABBa_K5300014) from the pRJPaph-mChe plasmid and designed the sgRNAs. We screened the loci from the genome, and amplified UP-feo-suicide [(BBa\_K5300027)](https://parts.igem.org/Part%3ABBa_K5300027) and DOWN-feo-suicide [(BBa\_K5300028)](https://parts.igem.org/Part%3ABBa_K5300028) by PCR. Then we performed seamless cloning to link them to pJQ200SK [(BBa\_K5300034)](https://parts.igem.org/Part%3ABBa_K5300034) (Figure 4-1-1).

![](https://static.igem.wiki/teams/5300/part/new/4-1-1.png)

**Fig. 4-1-1 The model of suicide circuit. [(BBa\_K5300041)](https://parts.igem.org/Part%3ABBa_K5300041)**

The recombinant plasmid was transferred into *E. coli*, and the correct recombinant plasmid was verified by colony PCR.

### Triparental mating and validation

The verified correct colonies were inoculated into gentamicin-resistant LB liquid medium under shaking conditions and cultured, triparental mating was performed using Helper, *Sinorhizobium fredii* CCBAU45436, and two screenings were performed. The single colonies obtained from the second screening were subjected to colony PCR utilizing the universal primer M13F/R.

Colony PCR demonstrated that the plasmid successfully underwent homologous recombination with *Sinorhizobium fredii* CCBAU45436, and the suicide circuit was successfully introduced into chassis. It is noteworthy that the module showed a different phenotype from the wild type when cultured for more than 3 d after introduction, i.e., the colonies became transparent teardrop-shaped.

### Fluorescence intensity verification

We utilized its fluorescence expression intensity to verify whether the suicide module functioned properly. We set up four sets of experiment with high nitrogen and high oxygen, low nitrogen and high oxygen, high nitrogen and low oxygen, as well as low nitrogen and low oxygen for verification. Subsequently, we used M9 medium without nitrogen source,then added different concentrations of ammonium chloride to form nitrogen concentration gradient. We added petroleum jelly after boiling the medium to isolate medium against atmosphere, creating anaerobic conditions. The OD600 value of the bacterial solution was determined after a period of shaking and incubation, and the fluorescence intensity of the bacterial solution was determined using a fluorescence spectrophotometer (Figure 4-3-1).

![](https://static.igem.wiki/teams/5300/source/experiment/4-3-1.png)

**Fig. 4-3-1 Ratio of relative fluorescence intensity to OD600 value of bacterial solution.** (a) Hypoxia. (b) Hyperoxia. Student’s t-test, ns: no significant difference; \*, p-value < 0.05; \*\*, p-value&lt0.01 \*\*\*, p-value < 0.001.

In a low oxygen environment, different nitrogen concentrations significantly affect the expression intensity of fluorescent proteins. This is consistent with our design. When changed to high oxygen environment, the change of nitrogen concentration could not significantly affect the fluorescent intensity, which may be caused by the environmental instability due to the expression of toxin. At the same time, we can also find that the fluorescent intensity of the bacterial solution cultured in high oxygen environment was higher than that in low oxygen environment, which may be related to the concentration of the bacterial solution fluctuates more in high oxygen environment by the change of toxin.

### Change in OD600 value of bacterial solution

In order to further verify whether the circuit leads to the death of the bacteria, we selected the strain with suicide circuit plasmid with OD600 value of 1, added different concentrations of NH4Cl, and investigated the changes of OD600 values after same interval under hyperoxic conditions (Figure 4-4-1).

![](https://static.igem.wiki/teams/5300/source/experiment/4-4-1.png)

**Fig. 4-4-1 The OD600 value after different incubation time.**

Obviously, we found that after a period of time, the OD600 value of the bacterial solution with additional nitrogen source showed a decreasing trend. And the decreasing trend was more obvious with the increase of the concentration of nitrogen source. Under this experimental condition, it can be considered that our suicide circuit is regulated by nitrogen concentration and the overall pathway construction is successful.

### Phenotype validation

In the process of triparental mating, we found that the correct exchanged strains did not express significant trait changes in the medium used for the first screening, whereas the phenotype of hyaline colonies appeared in the medium used for the second screening. We believe that the suicide module played a role in causing the strain to accumulate too much toxin and die. We therefore re-cultured the strain with the suicide module in nitrogen-free M9 medium shaking conditions. The cultured bacteria were spread on TY solid medium to observe their growth (Figure 4-5-1).

![](https://static.igem.wiki/teams/5300/source/experiment/4-5-1.png)

**Fig. 4-5-1 The pictures of bacterial colony.** (a) Culture for three days. (b) Culture for five days.

The toxin-antitoxin system functions under high nitrogen and high oxygen conditions, and the excess toxin content leads to colony death. We were able to observe that colonies from three-day cultures showed a milky white color, while colonies from five-day cultures showed a transparent color. This is the most direct evidence that the suicide circuit was successfully constructed.

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