Loading...
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.
·AtMRP3-GUS、AtMRP3-GFP、NtNRAMP3-GFP
·PCR2-GUS、Ntccx2-GFP、NtNRAMP2-GFP、NtNRAMP5-GFP、NtNRAMP6-GFP、PCR2-GFP
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
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
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.
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.
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.
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.
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.
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
AtRCD1-GUS
AtSRO1-GUS
AtRCD1-GFP
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
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:
AtMRP3
PCR2
NtNRAMP2
NtNRAMP3
NtNRAMP5
NtNRAMP6
NtCCX2
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.
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
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.
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)
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:
AtMRP3
AtPCR2
NtCCX2
SINRAMP4
NtNRAMP2
NtNRAMP3
NtNRAMP5
NtNRAMP6
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:
GUS-PCR2
GFP-Ntccx2
GFP-NtNRAMP2
GFP-NtNRAMP3
GFP-NtNRAMP5
GFP-NtNRAMP6
GFP-PCR2
These plasmids were eventually successfully transferred into Agrobacterium.
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.
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.
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:
NtNRAMP2-GUS、NtNRAMP5-GUS、NtNRAMP6-GUS、NtCCX2-GUS
Our
team
members continue to construct the above promoter GFP vector for subsequent
verification
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 .
(7.21~ 8.25) Three times before and after the team members, the following three batches of homologous recombination successful carriers into soil Agrobacterium:
1、AtRCD1-GUS、AtSRO1-GUS、AtRCD1-GFP
2、AtMRP3-GUS、AtMRP3-GFP、NtNRAMP3-GFP
3、GUS-PCR2、GFP-Ntccx2、GFP-NtNRAMP2、GFP-NtNRAMP3、GFP-NtNRAMP5、GFP-NtNRAMP6、GFP-PCR2
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.
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
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:
| groups | NtNRAMP2 | NtNRAMP5 | NtNRAMP6 |
|---|---|---|---|
| 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 |
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.
