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Project Experiment Section and Results Overview

The main experimental part of this project is roughly divided into the following five parts according to the actual progress and results of each stage of the experiment.

1. From 6.13 to 7.3, design experimental technical routes and implementation plans, learn and explore experimental conditions for promoter amplification and carrier construction.
2. From 7.3 to 7.17, the first round of experimental tests successfully constructed 2 promoters with a total of 3 different recombinant vectors: AtRCD1-GUS, AtSRO1-GUS, AtRCD1-GFP.
3. From July 24th to August 20th, the second round of screening promoter vector construction experiments successfully constructed a total of 9 different vectors for 7 promoters.


During this period, we simultaneously carried out tobacco seedling cultivation, plant transient transformation experiment preparation, and laboratory safety preparation related to CdCl2 operation.

From August 10th to August 29th, based on the GUS staining experiment technology, the cadmium response characteristics of four promoters were successfully obtained: NtNRAMP2, NtNRAMP6, NtNRAMP5, and AtMRP3. We believe that three promoters can be used for luminescent pathway testing.

Laboratory organization, experimental review and summary

First round of promoter screening

Due to weak understanding of molecular biology-related professional vocabulary, the team members encountered great difficulties in searching for promoters. After screening the first batch of candidate promoters, the supervisor helped select seven promoters with functional verification experimental evidence.

Original experimental technical route and progress plan (expected to take one month for the whole process):

1. Extraction of whole genes from plants derived from promoters (half a day/species)

2. Primer amplification PCR and running glue (1 day)

3. Vector amplification (shaking bacteria) (1 day)

4. Plasmid extraction (half day)

5. Carrier enzyme cutting + glue running + purification (2 days)

6. Homologous recombination + conversion to E. coli + coating (half day)

7. Pick spot + bacterial solution PCR (verify whether the connection is successful) (1 day)

8. In charge of sending sequencing (receiving the return result about 3-4 days after placing the order).

9. Shake the plasmid + transform Agrobacterium (1 day)

10. Pick spot + bacterial solution PCR (big shake) (PCR + running glue) (1 day)

11. Collect the bacterial liquid, use the existing infection liquid, treat it in the dark for 2 hours, and then inject it. After 2 days, collect plant leaves, petals and other tissues, stain them with GUS staining reagent, decolorize them with decolorizing reagent, and take photos to observe and record the plant staining situation after the leaves turn white

Experimental process and data for the construction of the first round of promoter vectors:

1. Extraction of whole genes from plants derived from promoters (actual use, 7.3-7.4)

The team extracted tobacco ( Nb ) genomic DNA and maize genomic DNA
BGI provided tomato genomic DNA , Arabidopsis genomic DNA
Ben's tobacco DNA extraction concentration (using Qubit fluorescence quantifier , quantitative concentration unit: ng /μL) Compared with the quality control sample WT1, the concentrations of samples 1-8 extracted by the team members all meet the standard and can be used for the next experiment.

(Samples 1-8 are the results of the concentration determination of fresh tobacco leaves taken by the team members, ground with liquid nitrogen, and extracted with a Qubit fluorescence quantifier ; WT1 is the DNA sample concentration of tobacco leaves provided by Professor Wang Xiaojuan of Shenzhen Laboratory, used for horizontal quality control to evaluate the quality and availability of samples extracted by the team members)

2. Primer amplification PCR and running glue (actual use, 7.4~ 7.6)

The glue chart from top to bottom is: 1-2 holes, AtRCD1-GFP; 3-4 holes, AtRCD1-GUS; 5-6 holes, AtSRO1-GFP; 7-8 holes, AtSRO1-GUS; 9-10 holes, SINRAMP4-GFP; 11-12 holes, SINRAMP4-GUS; 13-14 holes, NaNRAMP5-GFP; 15-16 holes, NaNRAMP5-GUS.

3. Vector amplification and plasmid extraction (including plasmid extraction, re-extraction after activation of low-activity bacteria, 7.6-7.9) (the E. coli stock solution containing GUS and GFP plasmids is provided by BGI)

The first plasmid extraction was completed, the concentration of GFP carrier plasmid DNA reached the standard, but the concentration of GUS bacteria extracted plasmid DNA did not meet the standard. It was inferred that the activity of GUS bacterial solution was low, so it was re-activated and shaken for re-extraction.

4. Carrier enzyme cutting + glue running + glue recycling (7.9~ 7.10)

GUS obtained 4 strips, holes 1-4 on the left; GFP obtained 2 strips, from the left, holes 5-6; the rubber strip was bright and the strip was clear; the above products were cut and recovered for subsequent carrier construction.

5. homologous recombination (after recombination into E. coli + coating + pick spot shaking bacteria + bacterial solution PCR ) (7.12~ 7.17)

On July 14th, the first round of homologous recombination , the first bacterial solution PCR results, no obvious bacterial P band, so the second round of homologous recombination experiments began.
On July 17th, the Escherichia coli plaque obtained by the second round of homologous recombination was subjected to spot shaking and bacterial liquid PCR , and it was found that each of the following three promoter vector combinations had 2 tubes of bacteria P positive, DP1, DP2 (AtRCD1-GFP first and second tubes positive), DS1, DS2 (AtRCD1-GUS first and second tubes positive)

OS1, OS2 (AtSRO1-GUS first and second tubes positive), perform secondary bacterial P verification on the above several tubes of bacterial solution, see the following gel chart.The results show that DP1 and DP2 are AtRCD1-GFP carriers, and the stripes are correct.
DS1 and DS2 construct result graphs for AtRCD1-GUS carriers, and the band situation is unknown.
OS1 and OS2 build result graphs for AtSRO1-GUS vectors with correct stripes.
We will send the above 6 tubes of bacterial liquid for sequencing, return the sequencing results, and let the supervisor help to view the map to confirm the successful construction of the vector.

Summary of the first round of carrier construction experiments

After exploring the experimental conditions in mid-June and conducting a two-week vector construction experiment from July 3rd to July 17th, the first round of 7 promoters, 14 combinations, and a total of 2 promoters and 3 different combinations of vectors were successfully constructed


On July 21st, we conducted the first discussion and summary of the formal experiment of the first phase of the project. We reviewed and summarized the reasons that may have led to the unsuccessful construction of several other promoters and combination carriers, including:

1. Some promoter primers bind to Ben's tobacco DNA template for low amplification efficiency, resulting in unsuccessful amplification
2. In the tomato promoter experiment, some team members were not familiar with the experimental operation at the beginning and added the wrong reagent in the parallel experiment
3. The promoter of Arabidopsis has the highest success rate in vector construction overall, but the team's aseptic operation is not strict, which may be the reason for the failure of one of the combination vectors

At the same time, we believe that if you want to light up tobacco, there is a high risk of failure with only the above two promoters, and we have launched the second round of promoter research

The second round of promoter research and screening

Based on the understanding of gene components in the first round of experiments, we began the second round of promoter screening. We found more than 20 promoters with cadmium response descriptions from relevant literature. After discussion and detailed literature review, we identified 8 promoters with strong evidence support, of which 2 promoters: AtMRP3 and AtPCR2 were independently investigated and demonstrated by the team members, and the other 6 promoters were searched and evaluated by the team members with the assistance of the supervisor, and finally confirmed to be selected for the second batch of experiments.

The following is a list of 8 promoters in the second round of promoter vector construction experiments:


Among them, PCR2 is independently designed by the team members, and the primers of the other promoters are designed by the mentor to guide the team members with tools such as snapgene and primerblast that come with the NCBI database, and after being checked and confirmed by the mentor, they are submitted to Shanghai Shenggong to help with primer synthesis and sent back.

Experimental process and data for the construction of the second round of promoter vectors:

1. Nt tobacco (K326) genomic DNA extraction (tobacco leaves from BGI)

Standard #2 Concentration: 10.0 ng /µl
Sample number: 1 Concentration: 16.2 ng /µl = 81.3 ng/ ml
Sample number: 2 Concentration: 17.5 ng /µl = 86.7 ng/ ml
Sample number: 3 Concentration: 18.7 ng /µl = 93.4 ng/ ml

The above experimental results showed that the DNA extraction concentration of 3 tubes of Nt tobacco samples reached the usable concentration

2. The second round of promoter primer amplification PCR and running glue

In the second round of experiments, after repeated PCR , we amplified a total of 16 products from 8 promoters combined with GUS and GFP.

The first 20 μL amplification system gel chart, each team member is responsible for their own promoter and the corresponding primer of the PCR amplification experiment, NtNRAMP5, AtMRP3, NtNRAMP3, NtNRAMP6 two carriers were amplified with obvious bands, NtCCX2 There are weak bands, indicating that among the 8 new promoters, 6 promoter primers were successfully designed; the next step will expand the amplification reaction system to 50 μL.

The first 50 μL amplification system gel diagram, several promoters that were successfully amplified in the previous sequence were unsuccessful in this round of amplification; however, the newly added NtNRAMP2-GUS has obvious bands.

We reviewed the results of this experiment and inferred that during the 50 μL amplification process, some team members operated the sample tube experiment for too long at room temperature before the PCR instrument , which affected the amplification enzyme activity and led to high primer dimer content. The product was not successfully amplified. We improved the experimental conditions and prepared all reactions to be transferred to the ice box for further amplification.

Due to untimely recording, the corresponding relationship between the gel chart and the sample is missing, but it can be seen that except for holes 1, 2, and 11, other products have clear stripes.

After the product of the above multiple PCR was purified, it was used for subsequent vector construction experiments.

3. homologous recombination and plaque growth (after recombination into E. coli + coating + pick spot shaking bacteria + bacterial solution PCR )

First homologous recombination (7.27~ 7.28)

For the first time, the remaining 6 promoters except PCR2 were subjected to homologous recombination . The above is the growth chart of E. coli. During this round of recombination experiments, due to the discovery of liquid culture medium contamination before use, no liquid culture medium was available. Therefore, after the recombination was completed, the recombinant bacteria were not revived and directly coated, resulting in poor plaque growth. Only CCX2-GUS (X2S), CCX2-GFP (X2P), NtNRAMP2-GFP ( P2P ), and NtNRAMP3-GFP (P13) had a small amount of plaque growth

Second homologous recombination (7.28~ 7.29)

NtNRAMP3 promoter, F13 is the culture dish after NtNRAMP3-GUS recombinant Escherichia coli coating; P13 is the culture dish after NtNRAMP3-GFP recombinant Escherichia coli coating

SINRAMP4 promoter, SS is SINRAMP4-GUS recombinant E. coli coated culture dish; SP is SINRAMP4-GFP recombinant E. coli coated culture dish

NtCCX2 promoter, X2S is the culture dish after NtCCX2-GUS recombinant Escherichia coli coating; X2P is the culture dish after NtCCX2-GFP recombinant Escherichia coli coating

NtRAMP5 promoter, P5S is the culture dish after NtRAMP5-GUS recombinant Escherichia coli coating; P5P is the culture dish after NtRAMP5-GFP recombinant Escherichia coli coating

NtRAMP2 promoter, P2P for NtRAMP2-GFP recombinant Escherichia coli coated dish; NtRAMP6 promoter, P6S is NtRAMP6-GUS recombinant Escherichia coli coated culture dish

AtMRP3 promoter, MRP3-S is the culture dish after AtMRP3-GUS recombinant Escherichia coli coating; MRP3-P is the culture dish after AtMRP3-GFP recombinant Escherichia coli coating

A total of 7 promoters, 11 carrier combinations, good plaque growth, can be used for spot-picking and shaking bacteria; the remaining P5P (NtRAMP5-GFP) needs to be re-coated and spot-picking and shaking bacteria
The above two rounds of E. coli bacterial solution PCR had no positive bands. The reason for the review may be that the recovery bacterial solution was centrifuged for a long time before coating, resulting in the destruction of the recombinant strain.

After optimizing the experimental steps, from early August to the end of August, we conducted multiple rounds of homologous recombination experiments for different carriers of GUS and GFP of the following promoters:


Among them, AtMRP3-GUS, AtMRP3-GFP, and NtNRAMP3-GFP carriers were successfully constructed, and there were no positive results for other promoters and carrier combinations.
AtMRP3-GUS, AtMRP3-GFP Escherichia coli homologous recombination, soil agrobacterium transformation and other experiments were successfully completed independently by team member Yao Youran.
The remaining promoters are completed by multiple team members in addition to the designated responsible team members
In the experiment, the operation of the high-pressure sterilization pot was operated by the experimental supervisor. The supervisor helped the team members to sterilize the culture solution and prepare other sterile operation experimental reagents and consumables, and accompanied the students throughout the operation of the ultra-clean workbench to ensure that the team members completed the experiment according to the aseptic operation essentials.
Six promoters were successfully recombined with seven different carriers, including:


These plasmids were eventually successfully transferred into Agrobacterium.

The following is a partial data chart of the homologous recombination experiment process

7.30~ 8.2 Homologous recombination experimental results

Promoter GUS-Ntccx2, GUS-NtNRAMP5, GUS-NtNRAMP6, GFP-NtNRAMP2, GFP-NtNRAMP3, GFP-NtNRAMP5, GFP-NtNRAMP6, GFP-Ntccx2 homologous recombination results showed that GUS-NtNRAMP5, GUS-NtNRAMP6 may be successful homologous recombination, bacterial P results showed positive strains, but the sequencing results were incorrect.

GUS-NtNRAMP5 sequencing result comparison: (using snapgene software to compare sequencing results by the supervisor)

Comparison of GUS-NtNRAMP6 sequencing results:

8.2~ 8.5 homologous recombination experimental results

Promoter GUS-Ntccx2, GUS-NtNRAMP3, GFP-NtNRAMP2, GFP-NtNRAMP3, GFP-NtNRAMP6, GFP-Ntccx2 homologous recombination glue chart results show that GUS-NtNRAMP3, GFP-NtNRAMP3 may be homologous recombination success, fungus p result band is correct, send sequencing (number GFP-NtNRAMP3-1, GFP (13) -NtNRAMP3-9, GFP (13) -NtNRAMP3-10, Gus (13) -NtNRAMP3-7, Gus (13) -NtNRAMP3-10). The sequencing results showed that GFP (13) -NtNRAMP3-10 was sequenced correctly, GFP (13) -NtNRAMP3-9 was bimodal, and the sequencing results of GFP-NtNRAMP3-1, GUS (13) -NtNRAMP3-7, and GUS (13) -NtNRAMP3-10 showed no load.

Comparison of sequencing results between GUS (13) -NtNRAMP3-7 and GUS (13) -NtNRAMP3-10:

Sequencing the bimodal bacterial solution, re-plate the line, pick a single colony, shake the bacteria, and send three tubes of bacterial solution for sequencing.

The above sequencing results are correct and empty bacterial liquid was shaken to extract the plasmid , and the electrophoresis results are as follows:

The above multiple rounds of experimental homologous recombination success rate is not high, we can not investigate to determine the cause, so after summarizing the results of multiple homologous recombination experiments, we sought guidance from Professor Wang Xiaojuan of Huada Research Institute, and hoped to get her help, so that we can get the target promoter as soon as possible.

Teacher Wang Xiaojuan helped us conduct a systematic review, suspecting that it was caused by the impurity of the recombinant experimental materials: promoter amplification sequence and the carrier itself after enzyme digestion. Therefore, the above two parts of the experimental materials were subjected to secondary purification before being put into use. The experimental time was tight, and the purification of the experimental materials was completed by the supervisor working overtime. The purified materials were handed over to some of our team members for further homologous recombination experiments.
At the same time, Teacher Xiaojuan helped to synchronize the construction of the GUS vector for the key experimental test promoter that we will use for subsequent experiments:


Our team members continue to construct the above promoter GFP vector for subsequent verification

The last round of homologous reorganization in August (8.15~ 8.20)

Promoter GUS-PCR2, GFP-Ntccx2, GFP-NtNRAMP2, GFP-NtNRAMP3, GFP-NtNRAMP5, GFP-NtNRAMP6, GFP-PCR2 homologous recombination results The electrophoresis results are shown in the figure below. Each group of bacterial liquids was selected from three tubes for testing. The sequencing results showed that the homologous recombination of the tested bacterial liquids was all successful. The correctly sequenced bacterial liquids were extracted by shaking plasmids .

4. Soil Agrobacterium transformation experiment

(7.21~ 8.25) Three times before and after the team members, the following three batches of homologous recombination successful carriers into soil Agrobacterium:


The following are the results of some soil Agrobacterium transformation experiments

After extracting the plasmid of the recombinant strain, it was transferred to Agrobacterium soils, coated and cultured at 28 ° C for 2 days, and a single colony was picked and shaken for bacterial p. The enzyme used for the first bacterial p did not amplify the band. After changing the high fidelity enzyme, the electrophoresis band size of promoter GFP-NtNRAMP3 was correct.

8.25~ 8.28 Agrobacterium transformation experiment

After the last round of homologous recombination was successful, the successfully constructed recombinant vector was transformed into Agrobacterium soil. the extracted plasmids GUS-PCR2, GFP-Ntccx2, GFP-NtNRAMP2, GFP-NtNRAMP3, GFP-NtNRAMP5, GFP-NtNRAMP6, GFP-PCR2 were transferred into Agrobacterium soil. coated and cultured at 28 ℃ for 2 days, and a single colony was picked and shaken for bacteria p. Electrophoresis showed successful transformation of Agrobacterium. The successfully transformed Agrobacterium was shaken and glycerol was used to preserve the bacteria.

5. Plant cadmium response transient transformation experiment

After seeking help from Teacher Wang Xiaojuan to construct the carrier, we obtained four recombinant carrier experimental materials: NtNRAMP2-GUS, NtNRAMP5-GUS, NtNRAMP6-GUS, and NtCCX2-GUS on August 11th. Combined with the tobacco cadmium response instantaneous transformation experiment we designed earlier, we carried out instantaneous transformation design and verification

Periwinkle Instantaneous Test

Because the tobacco seedlings cultivated in the laboratory are very valuable, we purchased periwinkle seedlings online in advance to ensure that the team members fully mastered the technical essentials of instantaneous rotation, and used periwinkle petals for instantaneous injection. The following are the test results of periwinkle instantaneous rotation

The potted plants of Changchun flowers purchased online were pretreated before the experiment and used for bacterial injection

Instantaneous transformation test results of periwinkle

Among them, PBS is a blank control, GUS-Super promoter is a positive control, and AtRCD1-promoter is one of our test promoters.

From the results of the petal instantaneous rotation experiment, the blank control was not stained blue, and the positive control was stained blue, indicating that the team members successfully injected the soil pus bacteria solution containing a strong promoter into the Changchun petals, completing the infection; at the same time, the blank control was not stained blue, indicating that there was no obvious pollution in our experimental operation. The test promoter AtRCD1-promoter did not show a blue reaction, indicating that the promoter, based on this instantaneous rotation exp ethod, cannot be expressed in Changchun petals.

Design of tobacco instantaneous experiment scheme

We designed the ideal experiment according to the mind map

However, we did not have enough tobacco seedlings that met the conditions for instantaneous injection, so we simplified the experimental plan based on laboratory conditions and finally obtained the following experimental results:

clean water
Cadmium (50ng/μL)
blank comparison
positive control
Conclusion Strong response, strong specificity Strongly responsive, weakly specific,
this promoter may be responsive to
drought in addition to cadmium
Strong response, strong specificity

Experimental summary

After conducting experiments in multiple stages, we believe that in a limited time, we have screened out many very promising plant cadmium response regulatory switches, such as NtNRAMP2, NtNRAMP6, and NtNRAMP5.

Due to time constraints, we cannot continue to conduct more functional verification experiments for promoters.

We hope that after the start of the school year, our team members will have more opportunities to explore and expand the experimental work in our project design, and ultimately, we can use the promoter components we have selected for plant cadmium response luminescence-related testing.

At the same time, we also requested help from the teachers at the Huada Research Institute. As the experimental difficulty of reconstructing the luminescent carrier itself is greater, we hope that they can conduct subsequent luminescence tests based on our existing experimental results.

At the same time, we also welcome more colleagues who are interested in this project to participate and explore with us on the basis of existing experimental results. We are willing to share all the experimental experience, experimental results, and component materials we can provide.

Next, our goal is to connect the screened cadmium response promoter switch to the luminescent gene pathway, so that the luminescent gene can only be expressed when there is cadmium, and the luminescent pathway works when there is cadmium, so that the plant can be bright, and then the luminescent plant can be used to monitor soil heavy metals.