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December
Week of December 20th to 26th
The previous team members were worked through two days of interviews and 16 members of Tsinghua igem 2022 were selected! Relay baton is now in our hands!Week of December 27th to January 2nd
Tsinghua iGEM 2022 had its first meeting and it was indeed awkward, maybe we didn't recognize each other clearly.
January
Week of January 3rd to 9th
Winter break started and we had our first meeting offline. The Tsinghua iGEM 2021 leaders explained the arrangements for winter break and everyone was still very ignorant about iGEM.
Week of January 10th to 16th
In order to grasp the rules of the iGEM competition initially，our assignment for the first week was to select an iGEM project from previous years for research and study, and to complete a 20min presentation in groups of four.
Week of January 17th to 23th
The second week's assignment was to regroup and choose a track conduct research.
Week of January 24th to 30th
This week's assignment was to do research on special awards, and a dozen special awards were divided into four topics. Still regrouping.
Week of January 31th to February 6th
This week's assignment is to read the iGEM 2021 judging book, which is the last assignment for our winter break!
Since we found out in last week's presentation that the education part can be done long before the topic of the project is determined, we formed a group this week to work on the science education videos. February
Week of February 7th to 13th
During these two weeks, we divided into four groups for brainstorm and presented the projects we wanted to work on in iGEM this year. This is the checkpoint of our brainstorm 1.
Week of February 14th to 20th and Week of February 21st to 27th
This week was checkpoint 2, where we refined and improved the projects presented last week based on the suggestions from other team members in the meeting. We each scored the projects of each group.
Week of February 28th to March 6th
We regrouped and started the brainstorm for the second round.
March
Week of March 7th to 13th and Week of March 14th to 20th
We regrouped and started the brainstorm for the second round.
Week of March 21st to 27th
As with the first round of brainstorm, this week was a time of iteration for the project.After the presentation, we scored again. The scores of the eight ideas from the two brainstorm rounds were sorted together and we will choose the two groups with the highest scores for pre-experimentation.
Although other ideas were not chosen, we are still impressed by the creativity behind these ideas.
Week of March 28th to April 3rd
It was the first week of the pre-lab, and each of our two groups presented their plans for the next month-long pre-experiments at meeting. Although there would be no more battles or choices to make this week, everybody was still very nervous.April
Week of April 4th to 10th
This is the time we got to our midterm week, everyone was a bit busy and there was no group meeting this week.Week of April 11th to 17th
Affibody-Pmr: the fusion protein sequence has been designed and we have ordered it in the gene synthesis platform.Nisin: we tried to obtain the sequence of nisA by pcr with short primers, but failed and it seems that we still need to purchases it.
We obtained Lactobacillus from the previous igem teams and performed colony PCR of 16SrRNA to verify.We had our new member in iGEM 2022: a pillow that is indeed so long. we call it worm dog.
Week of April 18th to 24th
Affibody-Pmr: We obtained EGFR fragments from plasmids in iGEM 2021.Nisin: We asked iGEM 2021 players how we should choose vectors and identified pET28a and pET32a as the governing vectors and started to design a cloning strategy.
Week of April 25th to May 1st
We're on Labor Day holiday this week, not much progress, and we had a big clean-up, let's all get some rest!May
Week of May 2nd to 8th
Nisin: This week we finished the construction and transformation of the experimental plasmids.Also, during the experiment we realized that Hifi is efficient as a means to construct clones, and the protocol and system of the experiment are gradually fixed and standardized.
Week of May 9th to 15th
This week we had our final presentation of two projects and we are gonna choose the projects we will be working on in iGEM2022 by secret ballot.Before the presentation took place, we had a captain's election.
Since it was a life and death week, our experiments were very heartfelt. Our biggest concern: whether or not the two-component system would work in E. coli, finally came to fruition this week. At this point we were still very new to the induction of the two-component system, and we were just starting to experiment with the induction time, the amount of induce molecule, and the incubation temperature. Also, fluorescence was not as easy to observe as we previously thought, and team members in iGEM 2021 taught us how to use the fluorescence microscope. We still remember the night when we were in the fluorescence microscope classroom observing the fluorescent bacteria with great anxiety, but the light of the slow flowing fluorescent bacteria greatly soothed our mood.
Thanks to our positive results, the sperm selection finally won in the pre-experiment stage!
Week of May 16th to 22th
As we approached the end of the semester, we divided our team members into groups , arranged the tasks that needed to be completed before the beginning of the summer.Week of May 23th to 29th
Unfortunately we are going to be in quarantine at school, and we have to decide whether we are gonna stay in school or go home. it is about how many people there will be here for wetlab in summer break. We had a vote.On the meeting this week, our PI Li Peng had us team training. We are touched and felt even closer to each other.
Week of May 30th to June 5th
And we held a picnic in our campus. We love pizza.June
Week of June 6th to 12th
One of our leader in iGEM2021 is going to leave school and studying abroad. We gathered together to take picture with him. (Love you World!)Week of June 13th to 19th
This is the first week of our summer vacation, before this week started; we had a summer pep rally to sort out our progress in wetlab, drylab and HP, and put forward our plans in the summer; we invited the leader of Tsinghua iGEM 2021 to share our experience on how to plan our summer vacation; finally, each of our team members shared their vision when first joined in iGEM 2022 , hoping to keep our project improving during the summer.We wrote our Impact Grant application this week and invited our Tsinghua iGEM 2019 team captain, Huang Zikang, to give us some advices and hopes that we would be successful in winning the award! (And it did!)
In terms of lab management, we organized the -20° refrigerator, so we finally don't have to rummage through boxes to find things; at the same time, we standardized the format of experiment records on benchling, and pre-saved several templates for common experiments, so perhaps it will be more convenient to record experiments.
Week of June 20th to 26th
Pmr and Amplifier
Week Plan:
1. Plasmid construction of pET28a-AffiPmrB-aa.2. Plasmid construction of pET28a-AffiPmrB-NEU.
3. Preliminary verification of AffiPmrB-A-C system.
Progress:
1. Plasmid construction of pET28a-AffiPmrB-aa.We prepared PCR fragments for HiFi assembly.
2. Plasmid construction of pET28a-AffiPmrB-NEU.
We successfully constructed this plasmid.
3. Preliminary verification of AffiPmrB-A-C system.
We did verification by fluorescence microscope.
Troubleshooting:
1. Plasmid construction of pET28a-AffiPmrB-aa.2. Plasmid construction of pET28a-AffiPmrB-NEU.
3. Preliminary verification of AffiPmrB-A-C system.
Problem:
The fluorescence intensity was too low.
Solution:
We plan to change our induction protocol and use other methods for verification.
Future Plan:
1. Plasmid construction of pET28a-AffiPmrB-aa.2. Verification of AffiPmrB-A-C system.
Nisin
Week Plan:
1. Plasmid construction of pNisAB and pNisAB(-)which contains NisA instead of NisA-affi
2. Plasmid construction of pNisC
3. Plasmid construction of pNisinOE2
Progress:
1. Plasmid construction of pNisAB and pNisAB(-):We have done the Hifi assembly.
2. Plasmid construction of pNisC:
We successfully constructed this plasmid.
3. Plasmid construction of pNisinOE2:
We successfully constructed this plasmid.
Future Plan:
1. Complete plasmid construction of pNisAB and pNisAB(-)2. Cotransform the pNisAB and pNisC to BL21(DE3)
3. Verify the expression of NisA-affi
4. Transform the pNisinOE2 to BL21(DE3)
5. Induce lacZ expression of pNisinOE2 with 1ng/ml nisin
Logic Gate
Week Plan:
1. Plasmid construction of v13. pET28a-ser integrase2. Plasmid construction of v19. pUC-mCherry-attB-OR-attP-EGFP
Progress:
1. Plasmid construction of v13. pET28a-ser integrase:We failed to construct this plasmid.
2. Plasmid construction of v19. pUC-mCherry-attB-OR-attP-EGFP:
We failed to insert mCherry fragment into the pUC vector containing the recombination sites of serine integrase.
Troubleshooting:
1. Cro&cI function verification:Problem:
Under the optimal induction condition, cI and cro both seemed to function. However, cro seem to be less effective than the positive control.
Analysis:
Conclusions were too soon to be drawn before we repeated the experiment for several times.
Solution:
We planned to repeat this experiment.
2. qPCR result:
Problem:
No significant result can be seen for qPCR even though microplate reading indicated stark contrast of fluorescence intensity before and after the induction. Several wells even showed no ct after qPCR, yet correct bands can be seen after we ran the qPCR product on the gel.
Analysis:
We speculated that our qPCR system was somewhat inappropriate.
Solution:
We planned to optimize our qPCR protocol and system.
Future Plan:
1. Cro&cI function verification: microplate reading to test new cro2. Serine integrase function verification: qPCR
HP
It was the first week of the summer vacation and we were about to start our HP. We contacted Ms. Lan Xie, whom we had consulted when we first decided the topic. She was teaching a course on reproductive health at Tsinghua University and had done some research on sperm quality test. At this time, we did not know much about current hospital sperm test and were still looking for a breakthrough point that we could target. Through our conversation with her, we learned about the current sperm testing methods in the hospital. She also provided constructive comments on our project.Others
This week, our artwork girl sorted out the painting tasks expected to be completed during the summer holidays.
(The comic she drew was so cute!!)The preliminary design of the Modeling section was also started this week, giving the output of the fluorescent protein under different starting protein concentration conditions.
Week of June 27th to July 1st
Pmr and Amplifier
Week Plan:
1. Plasmid construction of pET28a-AffiPmrB-aa.2. Verification of AffiPmrB-A-C system.
Progress:
1. Plasmid construction of pET28a-AffiPmrB-aa.We failed to construct the plasmid.
2. Verification of AffiPmrB-A-C system.
We did verification of the system by confocal.
Troubleshooting:
1. Plasmid construction of pET28a-AffiPmrB-aa.Problem:
The one-step-clone showed positive results by PCR, but sequencing showed negative results instead.
Analysis:
We found that the PCR primers we used to do PCR tests were somehow contaminated by template DNA, therefore the PCR couldn't screen out the positive clone.
Solution:
We changed all our primers and fragments for clone.
2. Verification of AffiPmrB-A-C system.
Nisin
Week Plan:
1. Complete plasmid construction of pNisAB and pNisAB(-)2. Cotransform the pNisAB and pNisC to BL21(DE3)
3. Verify the expression of NisA-affi
4. Transform the pNisinOE2 to BL21(DE3)
5. Induce lacZ expression of pNisinOE2 with 1ng/ml nisin
Progress:
1. Complete plasmid construction of pNisAB and pNisAB(-):We successfully constructed this plasmid.
2. Cotransform the pNisAB and pNisC to BL21(DE3)
The transformation is successful, but the efficiency is very low. Only one colony grew in the two plates.
3. Verify the expression of NisA-affi
Be limited by cotransformation efficiency, the work didn't begin this week.
4. Transform the pNisinOE2 to BL21(DE3)
We successfully tranform the plasmid to BL21(DE3).
5. Induce lacZ expression of pNisinOE2 with 1ng/ml nisin
The colonies were all blues whether it was induced by nisin or not.
Troubleshooting:
1. Low transformation efficiencyProblem:
The transformation is successful, but the efficiency is very low. Only one colony grew in the two plates.
Analysis:
Maybe the concentration of antibiotics in the solid medium is too high which inhibits the bacteria to grow.
Solution:
We planned to reduce the concentration of anbibiotics in the solid medium. We also planned to do cotransformation with electric-transformation method.
2. Failure of inducing expression of lacZ
Problem:
The colonies were all blues whether it was induced by nisin or not.
Analysis:
BL21(DE3) has endogenous lacZ expression.
Solution:
We planned to knock out the endogenous lacZ gene of BL21(DE3) or find out E.coli strain which has not endogenous lacZ gene.
Future Plan:
1. Cotransformation of pNisAB and pNisC: continue2. Verify the expression of NisA-affi
Logic Gate
Week Plan:
1. Plasmid construction of v13. pET28a-ser integrase2. Plasmid construction of v19. pUC-mCherry-attB-OR-attP-EGFP
Progress:
1. Plasmid construction of v13. pET28a-ser integrase:We successfully constructed this plasmid.
2. Plasmid construction of v19.
pUC-mCherry-attB-OR-attP-EGFP
We successfully inserted EGFP fragment into pUC vector containing the recombination sites of serine integrase, but still failed to insert mCherry fragment.
Troubleshooting:
Problem:The PCR result of linearization always showed ladder-like bands, resulting in low concentration after gel extraction.
Analysis:
We speculated unspecific binding of the primers to the template.
Solution:
We planned to redesign the primers to enhance specificity in the hope of a better linearization result.
Future Plan:
1. Plasmid construction of v19.
pUC-mCherry-attB-OR-attP-EGFP: continue
2. Serine integrase function verification: preliminary experiment
HP
This week we reached out to Dr. Yiqun Gu of the National Sperm Bank, a researcher studying male reproductive health. Two of our members met with him face-to-face. During our conversation with him, we learned more about the current hospital sperm test and the drawbacks of the current home sperm test strips. He recognized the innovative nature of the parts used in our project, but he suggested that our project still needed to consider practicality and the need to reduce costs as well as be user-friendly.Education
This week, we had the idea of joining forces with teams from iGEM teams in China for a joint science education project during an exchange with JLU and ShangTech students in charge of education.Others
We revisited the entire project design this week and realized that the current design has the problem of long detection times. We discussed at the group meeting how to make changes to the project, even including reversing the entire project. The conclusion was to spend a little time in the following week brainstorming in groups and presenting at the group meeting the following week.July
Week of July 5th to 10th
Pmr and Amplifier
Week Plan:
1. Plasmid construction of pET28a-AffiPmrB-aa.2. Verification of AffiPmrB-A-C system by other methods like microplate reader.
Progress:
1. Plasmid construction of pET28a-AffiPmrB-aa.We failed to construct this plasmid.
2. Verification of AffiPmrB-A-C system by other methods like microplate reader.
We tried confocal again but didn't use microplate reader.
Troubleshooting:
1. Plasmid construction of pET28a-AffiPmrB-aa.Problem:
The PCR test of KanR-positive clone showed that they had a insertion fragment of about 1000bp or 2000bp instead of the correct length 3600bp.
Analysis:
We inferred that the enzyme restriction linearization didn't work well with our HiFi primer.
Solution:
We plan to change our HiFi primer and do the HiFi assembly again.
2. Verification of AffiPmrB-A-C system by other methods like microplate reader.
Problem:
The microplate reader which can test fluorescence was not available that week.
Future Plan:
We planned to pause our experiment for a week and take the time to re-evaluate our project and rearrange further experiments.Nisin
Week Plan:
1. Cotransformation of pNisAB and pNisC2. Verify the expression of NisA-affi
Progress:
1. Cotransformation of pNisAB and pNisC:We successfully cotransform the two plasmids and the efficiency cotransformation was developed significantly.
2. Verify the expression of NisA-affi:
We used Ni column to purify NisA at first and did WB to verify the effect of purification. The concentration of NisA was low and the stripe of it in WB was inapparent.
Troubleshooting:
Failure of NisA purification:Problem:
The concentration of NisA was low and the stripe of it in WB was inapparent.
Analysis:
Maybe the Ni column's ability of protein binding is insufficient.
Solution:
We planned to use new column to purify, we also planned to buy protein concentration column to concentrate the purified protein.
Future Plan:
We planned to pause our experiment for a week and take the time to re-evaluate our project and rearrange further experiments.Logic Gate
Week Plan:
1. Plasmid construction of v19. pUC-mCherry-attB-OR-attP-EGFP2. Serine integrase function verification: preliminary experiment
Progress:
1. Plasmid construction of v19. pUC-mCherry-attB-OR-attP-EGFP:After weeks of efforts, we finally constructed this plasmid.
2. Serine integrase function verification
We co-transformed v18. pUC-attB-OR-attP-EGFP and v13. pET28a-ser integrase into BL21(DE3) strain to test for serine integrase function under IPTG induction.
We designed a pair of primers in the same direction before inversion, which would be in opposite directions after inversion, amplifying the sequences in between. Using this pair of primers, we tested whether serine integrase inverted the sequences between the two recombination sites.
We also sent the bacteria solution before and after inversion to sequencing for further confirmation.
Troubleshooting:
1. Leakage of serine integrase:Problem:
The PCR result of sequences before and after inversion all turned out to be positive.
Analysis:
After communicating with Ba Fang, who had conducted related research on serine integrase and its recombination sites, we speculated that integrase had already been expressing right after transformation without the inducer due to leakage.
Solution:
Considering Ba Fang' s advice, we planned to examine the extent of leakage first in order to set a threshold for inversion verification. In other words, as long as stark contrast before and after the inversion can be established, the problem of leakage could be ignored.
2. Efficiency of inversion by serine integrase:
Problem:
The sequencing result of bacteria solution before and after inversion all showed that the sequences had NOT been inverted.
Analysis:
We speculated that sequences that had been inverted accounted for the vast majority, indicating that our induction was insufficient.
Solution:
We planned to optimize the induction condition in further experiments.
Future Plan:
We planned to pause our experiment for a week and take the time to re-evaluate our project and rearrange further experiments.
HP
When constructing a two-component downstream logic system, we were inspired by the article published by Jian Li's team in 2022 (doi: 10.1093/nar/gkac124) and decided to use tryptophan integrase as the main control element for logic gating. Initially, we used a tryptophan integrase synthesized by the company, however, it was less efficient. In order to improve the efficiency of the tryptophan integrase, we contacted Professor. Jian Li to obtain the plasmid expressing the tryptophan integrase and its target of action mentioned in the article. Through Professor. Li, we contacted the first author, Mr. Fang Ba. He was kind enough to send us the plasmid and gave us advice on the design and details of our experiments.Education and Communication
This week, our team members visited Bluepha and had face-to-face communication to further understand the current state of the synthetic biology industry.Others
This week, we split into four groups and presented ideas for possible changes to our current project. The final vote was to adopt the toehold switch, a more responsive design that would hopefully solve the problems of our project; during the discussion, everyone was very involved and it seemed like a nostalgic return to the brainstorm state of the early days of the team.Week of July 11th to 17th
Pmr and Amplifier
Week Plan:
We planned to pause our experiment for a week and take the time to re-evaluate our project and rearrange further experiments.Progress:
We further consulted with Ba Fang, and proposed several solutions to tackle with the problems in our experiments.Troubleshooting:
1. Efficiency of inversion by serine integrase:Problem:
The efficiency of inversion by serine integrase was too low to cause any difference before and after the induction.
Analysis:
We speculated that the low efficiency was due to improper induction condition or insufficient signal amplification.
Solution:
To be on the safe side, we changed part of our design and decided to use toehold switch-trigger RNA system as a possible substitute if the problem of serine integrase could not be solved.
2. Fluorescence intensity of AffiPmrB-A-C system:
Problem:
The fluorescence intensity of induced AffiPmrB-A-C system was too low, and was unstable showed by the fluorescence microscope.
Solution:
Verification again; try the variant AffiPmrB-aa; add amplifier to the system.
Future Plan:
1. Plasmid construction of pBAD24-T7T3-LacZ-Toehold Switch 1 & pBAD24-T7T3-LacZ-Toehold Switch 2.2. Plasmid construction of pET28a-mCherry-Toehold Switch 1 & pET28a-mCherry-Toehold Switch 2.
3. Construction of NisK/R-amplifier system.
4. Further verification of AffiPmrB-A-C system.
Nisin
Week Plan:
We planned to pause our experiment for a week and take the time to re-evaluate our project and rearrange further experiments.Progress:
We bought the protein concentration column. We decided to keep our original experiment plan after re-evaluation.
Future Plan:
1. Verify the expression of NisA-affi2. Use microplate reader to detect the expression of EGFP of pNisinOE1
Logic Gate
Week Plan:
We planned to pause our experiment for a week and take the time to re-evaluate our project and rearrange further experiments.Progress:
We further consulted with Ba Fang, and proposed several solutions to tackle with the problems in our experiments.
Troubleshooting:
1. Leakage of serine integrase:Problem:
Serine integrase started expressing even without induction due to leakage problem.
Analysis:
We speculated that the leakage may be reduced if we changed the expression and induction system.
Solution:
We planned to change to araC-pBAD system to express the serine integrase under arabinose induction given that araC-pBAD system had been proved of weak leakage in two-component system induction in previous work.
2. Efficiency of inversion by serine integrase:
Problem:
The efficiency of inversion by serine integrase was too low to cause any difference before and after the induction.
Analysis:
We speculated that the low efficiency was due to improper induction condition or insufficient signal amplification.
Solution:
We planned to try arabinose induction first. If failed, we planned to try to introduce a resource allocator or amplifier into our genetic circuit to further amplify the signal.
Future Plan:
1. Plasmid construction of v34. pBAD-ser integrase
2. Plasmid construction of v32. Final_mCherry-OR-EGFP-pBAD-araC-cro-cI
HP
This week we compiled our previous HP records, analyzed the suggestions made by our consultants, and began thinking about how to improve our program based on these suggestions. We have kept in touch with Mr. Fang Ba to continue to address the issues encountered on serine integrase.Education and Communication
This week we had the first workshop of TAP. In this workshop, the three teams gave an overview of their 2022 projects and discuss how the TAP teams can bridge the contexts of science and art to reflect and create together.Week of July 18th to 24th
Pmr and Amplifier
Week Plan:
1. Plasmid construction of pBAD24-T7T3-LacZ-Toehold Switch 1 & pBAD24-T7T3-LacZ-Toehold Switch 2.2. Plasmid construction of pET28a-mCherry-Toehold Switch 1 & pET28a-mCherry-Toehold Switch 2.
3. Further verification of AffiPmrB-A-C system.
Progress:
1. Plasmid construction of pBAD24-T7T3-LacZ-Toehold Switch 1 & pBAD24-T7T3-LacZ-Toehold Switch 2.We prepared PCR fragments for HiFi assembly, and sent the LacZ-Toehold Switch 1&2 sequence to company for synthesis.
2. Plasmid construction of pET28a-mCherry-Toehold Switch 1 & pET28a-mCherry-Toehold Switch 2.
We sent the mCherry-Toehold Switch 1&2 sequence to company for synthesis.
3. Further verification of AffiPmrB-A-C system.
We tested the EGFP fluorescence intensity of AffiPmrB-A-C system by microplate reader.
Troubleshooting:
1. Further verification of AffiPmrB-A-C system.Problem:
The fluorescence intensity of EGFP didn't change much after being induced, and was unstable.
Analysis:
The augmenter of EGFP expressed was too low that the error in measurement would cause unstable results, meanwhile the system itself may not be that stable.
Solution:
We plan to add amplifier to the AffiPmrB-A-C system.
Future Plan:
1. Plasmid construction of pBAD24-T7T3-LacZ-Toehold Switch 1 & pBAD24-T7T3-LacZ-Toehold Switch 2.2. Plasmid construction of pET28a-mCherry-Toehold Switch 1 & pET28a-mCherry-Toehold Switch 2.
3. Plasmid construction of pET28a-AffiPmrB-aa.
Nisin
Week Plan:
1. Verify the expression of NisA-affi2. Use microplate reader to detect the expression of EGFP of pNisinOE1
Progress:
1. Verify the expression of NisA-affiWe did the purification twice and the stripe was a little bit more apparent than last time.
2. Use microplate reader to detect the expression of EGFP of pNisinOE1
The expression of EGFP was low.
Troubleshooting:
Problem:The expression of EGFP was low.
Analysis:
IPTG inducement didn't work well.
Solution:
We planned to replace the T7 promoter into the constitutive promoter.
Future Plan:
1. Replace the T7 promoter into the constitutive promoter2. Use purified NisA to induce expression
Logic Gate
Week Plan:
1. Plasmid construction of v34. pBAD-ser integrase2. Plasmid construction of v32. Final_mCherry-OR-EGFP-pBAD-araC-cro-cI
3. Serine integrase function verification:qPCR
Progress:
1. Plasmid construction of v34. pBAD-ser integrase:We successfully constructed this plasmid.
2. Plasmid construction of v32. Final_mCherry-OR-EGFP-pBAD-araC-cro-cI:
We successfully inserted mCherry fragment and the new promoter OR into pET28a-EGFP vector (v30). We also constructed the fragment of pBAD-araC-cro (v31).
3. Serine integrase function verification:
We examined the inversion rate of serin integrase by qPCR under IPTG induction.
Troubleshooting:
1. Inversion efficiency of serine integraseProblem:
The ct value of the sample before and after induction differed by 3 ct, indicating that the concentration of inverted sequences was 8 times compared with the concentration of the original sequences.
Analysis:
Even though differences before and after induction did exist, but it was insufficient to establish any contrast on fluorescence.
Solution:
We planned to run qPCR again after constructing our new plasmid with pBAD promoter.
Future Plan:
1. Plasmid construction of v32. Final_mCherry-OR-EGFP-pBAD-araC-cro-cI: insert pBAD-araC-cro and cI fragments
2. Serine integrase function verification: co-transform v34. pBAD-ser integrase and v30. pET28a-mCherry-OR-EGFP
HP
We continued to improve serine integrase on the suggestions given by Mr. Fang Ba. In the meantime, we made preliminary design of the hardware and contacted relevant professors to ask for methods.Week of July 25th to 30th
Pmr and Amplifier
Week Plan:
1. Plasmid construction of pBAD24-T7T3-LacZ-Toehold Switch 1 & pBAD24-T7T3-LacZ-Toehold Switch 2.2. Plasmid construction of pET28a-mCherry-Toehold Switch 1 & pET28a-mCherry-Toehold Switch 2.
3. Plasmid construction of pET28a-AffiPmrB-aa.
Progress:
1. Plasmid construction of pBAD24-T7T3-LacZ-Toehold Switch 1 & pBAD24-T7T3-LacZ-Toehold Switch 2.
We successfully constructed the two plasmids.
2. Plasmid construction of pET28a-mCherry-Toehold Switch 1 & pET28a-mCherry-Toehold Switch 2.
The sequence sent for synthesis hadn't finished yet.
3. Plasmid construction of pET28a-AffiPmrB-aa.
We failed in constructing this plasmid.
Troubleshooting:
1. Plasmid construction of pET28a-AffiPmrB-aa.Problem:
The PCR test of KanR-positive clone still showed that they had a insertion fragment of about 1000bp or 2000bp instead of the correct length 3600bp.
Analysis:
We inferred that there might be other unexpected homologous sequence on the vector.
Solution:
We decided to change our clone strategies.
Future Plan:
1. Verification of LacZ-Toehold Switch 1&2.2. Plasmid construction of pET28a-mCherry-Toehold Switch 1 & pET28a-mCherry-Toehold Switch 2.
3. Plasmid construction of pET28a-AffiPmrB-aa.
Nisin
Week Plan:
1. Replace the T7 promoter into the constitutive promoter and construct the pNisin12. Use purified NisA to induce expression
3. Express the NisA-affi
Progress:
1. Replace the T7 promoter into the constitutive promoter:We successfully replaced the promoter.
2. Express the NisA-affi:
We successfully expressed the NisA-affi and purified it.
3. Use purified NisA to induce expression
We used gradient concentration of nisin to induce expression of pNisinOE1, but the curve of expression can't be repeated.
Troubleshooting:
Problem:Expression results can't be repeated
Analysis:
We used 12ml shaking tubes to do the expression, which made differences between groups, and the exact concentration of bacteria is different in each tube and each experiment.
Solution:
We planned to use conical flask to do the expression and detect the expression of EGFP by inducing it with a different time.
Future Plan:
1. Expression of pNisin12. Constuct the pNisin2
Logic Gate
Week Plan:
1. Plasmid construction of v32. Final_mCherry-OR-EGFP-pBAD-araC-cro-cI: insert pBAD-araC-cro and cI fragments2. Serine integrase function verification: co-transform v34. pBAD-ser integrase and v30. pET28a-mCherry-OR-EGFP
Progress:
1. Plasmid construction of v32. Final_mCherry-OR-EGFP-pBAD-araC-cro-cI:We inserted pBAD-araC-cro and cI fragments into the vector, and the final plasmid was sent to sequencing.
2. Serine integrase function verification:
The co-transformation of v34. pBAD-ser integrase and v30. pET28a-mCherry-OR-EGFP into BL21(DE3) strain failed.
Troubleshooting:
1. Failure of co-transformationProblem:
Several rounds of co-transformation all failed. No colonies can be seen on the plate.
Analysis:
Such results indicated that plasmids failed to be included into the bacteria during co-transformation.
Solution:
We planned to optimize the co-transformation system by adjusting the amount of plasmids being transformed.
Future Plan:
1. Plasmid construction of v32. Final plasmid: confirm sequencing result
2. Serine integrase function verification: test inversion rate by qPCR under arabinose induction
HP
We started with the overall design of the sperm test chip, but as the members were relatively new to hardware design and actual construction, we consulted Professor Chen, who has been an iGEM instructor for many years, for advice. During our consultation with Professor Guoqiang Chen, he mentioned that the cost of microfluidic chips might be an issue, and this is what we were reminded of during the hardware exchange with the BIT team: we should try to control the cost when designing microfluidic systems. In this regard, we approached Taiyan Zhou, who has extensive experience in microfluidic chip design, to ask him how to minimise costs.Education and Communication
This week we conducted the second workshop of TAP and invited four artists to share case studies of their artistic practices in the fields of bio-design, bio-art, and curation.Others
We started writing on our wiki this week.August
Week of August 1st to 7th
Pmr and Amplifier
Week Plan:
1. Verification of LacZ-Toehold Switch 1&2.2. Plasmid construction of pET28a-mCherry-Toehold Switch 1 & pET28a-mCherry-Toehold Switch 2.
Progress:
1. Verification of LacZ-Toehold Switch 1&2.We failed in verifying LacZ-Toehold Switch 1&2.
2. Plasmid construction of pET28a-mCherry-Toehold Switch 1 & pET28a-mCherry-Toehold Switch 2.
We successfully constructed the plasmid pET28a-mCherry-Toehold Switch 2, while the sequence mCherry-Toehold Switch 1 still hadn't been synthesized.
Troubleshooting:
1. Verification of LacZ-Toehold Switch 1&2.Problem:
Nothing changed after X-gal and arabinose had been added.
Analysis:
We inferred that we hadn't successfully induced the promoter pBAD.
Solution:
We planed to test pBAD first and build a protocol of pBAD inducing.
Future Plan:
1. Verification of LacZ-Toehold Switch 1&2.2. Plasmid construction of pET28a-mCherry-Toehold Switch 1.
3. Verification of mCherry-Toehold Switch 2.
Nisin
Week Plan:
1. Expression of pNisin12. Constuct the pNisin2
Progress:
1. Expression of pNisin1We transformed pNisin1 into DH5α and induced it with nisin, but the plates were all white. No expression of lacZ was detected.
2. Constuct the pNisin2
The stripes in the gel after colony PCR were weird.
Troubleshooting:
1. Failure of expression:Problem:
The plates were all white. No expression of lacZ was detected.
Analysis:
DH5α strain is not a good expression strain, the protein is not normally expressed or the expression amount is too little.
Solution:
We planned to replace lacZ into EGFP and use microreader to detect expression.
2. Colony PCR:
Problem:
The stripes in the gel after colony PCR were weird.
Analysis:
Maybe there is something wrong with the agarose gel we prepared.
Solution:
We send the plasmid to the company for sequencing.
Future Plan:
1. Replace lacZ into EGFP and use microplate reader to detect expression.2. Use NisA-affi to induce expression.
Logic Gate
Week Plan:
1. Plasmid construction of v32. Final plasmid: confirm sequencing result2. Serine integrase function verification: test inversion rate by qPCR under arabinose induction
Progress:
1. Plasmid construction of v32. Final plasmid:Sequencing results confirmed that our plasmid construction was successful.
2. Serine integrase function verification:
We co-transformed v34. pBAD-ser integrase and v30. pET28a-mCherry-OR-EGFP into BL21(DE3) strain. Colony PCR results showed that our co-transformation was successful.
We inducted under different conditions (arabinose concentration, temperature, and time) to explore the optimal experiment condition for serine integrase to function.
We performed qPCR after arabinose induction to test the inversion rate of serine integrase, yet no significant difference before and after induction can be seen.
Troubleshooting:
1. qPCR results:Problem:
No significant results can be seen before and after induction.
Analysis:
We speculated that the induction condition was still not optimal, thus insufficient amount of serine integrase can be expressed. In addition, the qPCR system was not rigorous as well.
Solution:
We planned to introduce an internal reference in qPCR.
Future Plan:
1. Serine integrase function verification: qPCR
2. Cro&cI function verification: fluorescence measurement by microplate reader
HP
As for software, what we want to do is essentially a regression problem, and one of the datasets found now is the promoter sequences of prokaryotes and the expression levels of their corresponding genes. We hope to be able to predict the corresponding intensity given the promoter sequence by using some deep learning models. So we asked Zheni Zeng, a PhD student in the Natural Language Processing and Social Humanities Computing Laboratory (THUNLP) at Tsinghua University, for advice.Education and Communication
This week we had the third workshop of TAP. CAFA_China presented their team's exhibition proposal and overall plan in detail, followed by a discussion and adjustments.Week of August 8th to 14th
Pmr and Amplifier
Week Plan:
1. Verification of LacZ-Toehold Switch 1&2.2. Plasmid construction of pET28a-mCherry-Toehold Switch 1.
3. Verification of mCherry-Toehold Switch 1&2.
Progress:
1. Verification of LacZ-Toehold Switch 1&2.We successfully tested pBAD.
2. Plasmid construction of pET28a-mCherry-Toehold Switch 1.
We successfully constructed this plasmid.
3. Verification of pET28a-mCherry-Toehold Switch 1&2.
We failed in inducing the trigger RNA.
Future Plan:
1. Verification of LacZ-Toehold Switch 1&2.2. Verification of mCherry-Toehold Switch 1&2.
3. Construction of plasmid J23100-NisK-NisR-PnisA-T7T3-EGFP.
Nisin
Week Plan:
Because all our team members are not at school, this week's experiment is suspended.Progress:
Future Plan:
1. Replace lacZ into EGFP and use microplate reader to detect expression.2. Use NisA-affi to induce expression.
Logic Gate
Week Plan:
1. Serine integrase function verification: qPCR2. Cro&cI function verification: fluorescence measurement by microplate reader
Progress:
1. Serine integrase function verification: qPCRWe co-transformed v34. pBAD-ser integrase and v30. pET28a-mCherry-OR-EGFP into BL21(DE3) strain. Colony PCR results showed that our co-transformation was successful.
We inducted under different conditions (arabinose concentration, temperature, and time) to explore the optimal experiment condition for serine integrase to function.
We performed qPCR after arabinose induction to test the inversion rate of serine integrase, yet no significant difference before and after induction can be seen.
2. Cro&cI function verification: fluorescence measurement by microplate reader
We transformed v32. Final plasmid into BL21(DE3) strain to test the function of cro and cI under arabinose induction.
We inducted under different conditions (arabinose concentration, temperature, and time) to explore the optimal experiment condition for cro and cI to function.
We measured fluorescence intensity by microplate reader after arabinose induction to test the function of cro and cI. cI seemed to function, yet cro had no significant result.
Troubleshooting:
1. qPCR result:Problem:
No significant result can be seen before and after induction. We ran the qPCR product on the gel, but nothing showed on the gel.
Analysis:
We speculated that either one or both of the plasmids were lost after co-transformation. Antibiotic was supposed to be selective, but those bacteria without chloramphenicol resistance could still survive on some residual chloramphenicol in the solution. Another possibility was that pBad promoter or araC was not working.
Solution:
To tackle the issue of losing plasmids after co-transformation, we planned to construct the serine integrase and its recombination sites on the same plasmid, namely plasmid v41. Two in one. To test whether the pBad promoter could work, we planned to design a verification plasmid to test the function of pBad promoter.
2. Cro&cI function:
Problem:
Under the optimal induction condition, cI seemed to function well, but cro has no significant result.
Analysis:
Conclusions were too soon to be drawn before we repeated the experiment for several times.
Solution:
We planned to repeat this experiment.
Future Plan:
1. Cro&cI function verification: fluorescence measurement by microplate reader
2. Plasmid construction of Two in one plasmid
3. Plasmid construction of the promoter verification plasmid
HP
Since we are making a product for the public, this week we published a questionnaire to ask potential users about their willingness to use it and the public acceptability of synthetic biology for home testing devices.Education and Communication
This week we had the fourth workshop of TAP, where our team presented our exhibition proposal and discussed with CAFA_China and Peking team.Others
We attended the offline meeting held by iBowu and were very happy to finally meet iGEMers from other teams off-line!Week of August 15th to 21st
Pmr and Amplifier
Week Plan:
1. Verification of LacZ-Toehold Switch 1&2.2. Verification of mCherry-Toehold Switch 1&2.
3. Construction of plasmid J23100-NisK-NisR-PnisA-T7T3-EGFP.
Progress:
1. Verification of LacZ-Toehold Switch 1&2.We failed in verifying LacZ-Toehold Switch 1&2.
2. Verification of mCherry-Toehold Switch 1&2.
We failed in verifying mCherry-Toehold Switch 1&2.
3. Construction of plasmid J23100-NisK-NisR-PnisA-T7T3-EGFP.
We prepared all PCR fragments for HiFi assembly.
Future Plan:
1. Verification of LacZ-Toehold Switch 1&2.2. Verification of pET28a-mCherry-Toehold Switch 1&2.
3. Construction of plasmid J23100-NisK-NisR-PnisA-T7T3-EGFP.
Nisin
Week Plan:
1. Replace lacZ into EGFP and use microplate reader to detect expression.2. Use NisA-affi to induce expression.
3. Knock out lacZ of BL21(DE3)
Progress:
1. Use NisA-affi to induce expression:We used Nisin and NisA-affi to induce expression for a lot of times, but the results had big moves, we coundn't get a stable result.
2. Replace lacZ into EGFP and use microplate reader to detect expression:
We successfully constructed the plasmid.
3. Knock out lacZ of BL21(DE3)
We bought the CRISPR-Cas9 system and designed four guide RNAs for CRISPR-Cas9.
Troubleshooting:
Problem:We used Nisin and NisA-affi to induce expression for a lot of times, but the results had big moves, we coundn't get a stable result.
Analysis:
Maybe the expression of EGFP was low which made microplate reader difficult to distinguish.
Solution:
We planned to use fluorescence microscope to observe EGFP expression directly
Future Plan:
1. Use NisA-affi to induce expression.2. Use fluorescence microscope to observe EGFP expression directly
3. Knock out lacZ of BL21(DE3)
Logic Gate
Week Plan:
1. Cro&cI function verification: fluorescence measurement by microplate reader2. Plasmid construction of Two in one plasmid
3. Plasmid construction of the promoter verification plasmid
Progress:
1. Cro&cI function verification: fluorescence measurement by microplate readerWe transformed v32. Final plasmid into BL21(DE3) strain to test the function of cro and cI under arabinose induction.
We inducted under different conditions (arabinose concentration, temperature, and time) to explore the optimal experiment condition for cro and cI to function.
We measured fluorescence intensity by microplate reader after arabinose induction to test the function of cro and cI. cI seemed to function well, yet cro had no significant result.
2. Plasmid construction of Two in one plasmid
We finished constructing v41. Two in one plasmid. Sequencing result confirmed that our construction was successful.
3. Plasmid construction of the promoter verification plasmid
We constructed v37. pBad-mCherry plasmid and v38. pBad-mCherryOR plasmid to test promoter function, but sequencing result failed.
Troubleshooting:
1. Cro&cI function:Problem:
Under the optimal induction condition, cI seemed to function well, but cro has no significant result.
Analysis:
One possibility was that the pBad promoter was not working. Another possibility was that the cro fragment was not working.
Solution:
We planned to test the pBad promoter function first.
2. Failed sequencing result of the promoter verification plasmid:
Problem:
The sequencing of the bacteria solution turned out to be positive, but the sequencing of plasmid failed due to low concentration.
Analysis:
The concentration of DNA was indeed very low after plasmid extraction.
Solution:
We planned to repeat the construction from HiFi and transformation.
Future Plan:
1. Serine integrase function verification: qPCR and microplate reading
2. Plasmid construction of the promoter verification plasmid
3. Promoter function verification
HP
We learned that Prof. Jinming Lin from Tsinghua University is dedicated to the research of microfluidics-based rapid microbial assays, including the development of instruments and kits related to the assay technology. In order to obtain more professional guidance on microfluidics, this week we contacted Prof. Jinming Lin via email. Professor Lin has a long history of teaching courses related to microfluidics and has extensive experience in guiding undergraduate students in their research.Education and Communication
This week we had the fourth workshop of TAP, this time it was the presentation of the exhibition of Peking team.Others
We were all very encouraged to learn that we had won three awards at CCiC!Week of August 22nd to 28th
Pmr and Amplifier
Week Plan:
1. Verification of LacZ-Toehold Switch 1&2.2. Verification of mCherry-Toehold Switch 1&2.
3. Construction of plasmid J23100-NisK-NisR-PnisA-T7T3-EGFP.
4. Verification of NisK/R-amplifier.
Progress:
1. Verification of LacZ-Toehold Switch 1&2.We failed in verifying LacZ-Toehold Switch 1&2.
2. Verification of mCherry-Toehold Switch 1&2.
We successfully verified mCherry-Toehold Switch 1&2.
3. Construction of plasmid J23100-NisK-NisR-PnisA-T7T3-EGFP.
We successfully constructed this plasmid.
4. Verification of NisK/R-amplifier.
We successfully verified NisK/R-amplifier by EGFP fluorescence intensity using fluorescence microscope.
Future Plan:
1. Verification of LacZ-Toehold Switch 1&2 (If it still remains no result, we would give up Toehold Switch).2. Change the promoter of Toehold Switch-mCherry for a stronger one.
3. Verification of NisK/R-amplifier.
Nisin
Week Plan:
1. Use NisA-affi to induce expression.2. Use fluorescence microscope to observe EGFP expression directly
3. Knock out lacZ of BL21(DE3)
Progress:
1. Use NisA-affi to induce expression.We used Nisin and NisA-affi to induce expression for a lot of times, but the results had big moves, we coundn't get a stable result.
2. Use fluorescence microscope to observe EGFP expression directly
We induced pNisin2 with 4 h and detected the fluorescence use flurescence microscope. The results were positive, but the induction effect was not ideal.
3. Knock out lacZ of BL21(DE3)
We failed to knock the gene, and we gave up due to the time limitation.
Troubleshooting:
1. The induction effect was not idealProblem:
We induced pNisin2 with 4 h and detected the fluorescence use flurescence microscope. The results were positive, but the induction effect was not ideal.
Analysis:
The PnisA promoter had relatively strong leakage, which made the induction effect inapparent.
Solution:
We planned to change the expression strain from BL21(DE3) to Mach1-T1, which was used widely for expression to improve the induction effect.
2. Failed to knock out the lacZ gene:
Problem:
We failed to knock the gene, and we gave up due to the time limitation.
3. Unstable expression curve
Problem:
We induced pNisin2 with 4 h and detected the fluorescence use flurescence microscope. The results were positive, but the induction effect was not ideal.
Analysis:
Maybe the NisK could not transfer to cell membrane well, which made it difficult to accept the signal of Nisin.
Solution:
We planned to construct a plasmid containing nisK which has EGFP downstream to observe the transfer of EGFP to cell membrane.
Future Plan:
1. Use NisA-affi to induce expression.2. Change the expression strain to Mach1-T1
3. Construct the plasmid containing NisK with EGFP downstream
Logic Gate
Week Plan:
1. Serine integrase function verification: qPCR and microplate reading2. Plasmid construction of the promoter verification plasmid
3. Promoter function verification
Progress:
1. Serine integrase function verification: qPCR and microplate readingWe transformed v41. Two in one plasmid into BL21(DE3) strain to test integrase function.
We inducted under various conditions (1% arabinose, different induction time, and different induction temperature) to explore the optimal induction condition for serine integrase to function.
We performed qPCR after arabinose induction to test the inversion rate of serine integrase, yet no significant difference before and after induction can be seen.
We also measured the fluorescence intensity using microplate reader to examine the inversion rate of serine integrase.
2. Plasmid construction of the promoter verification plasmid
We repeated the construction, and the sequencing result confirmed that our construction was successful. The two promoter verification plasmids (v37. & v38.) were ready for function verification.
3. Promoter function verification We transformed v37. pBad-mCherry plasmid into BL21(DE3) strain. After 6h of arabinose induction, the bacteria turned red.
Troubleshooting:
1. Serine integrase function verification:Problem:
The qPCR result showed that the difference between before and after induction was 1 ct at most. However, microplate reading showed significant contrast of fluorescence intensity before and after induction. We also discovered that the fluorescence intensity was already very high before induction at 16℃.
Analysis:
We speculated that our qPCR protocol might need to be improved. Apart from that, we speculated that there might be some leakage from pBad promoter or OR that resulted in the high background fluorescence level.
Solution:
We planned test whether there was any leakage from pBad promoter or OR using the promoter verification plasmid.
2. Cro&cI function:
Problem:
Under the optimal induction condition, cI seemed to function well, but cro has no significant result.
Analysis:
One possibility was that the pBad promoter was not working. Another possibility was that the cro fragment was not working.
Solution:
We planned to test the pBad promoter function first. If the promoter proved to be working, then we would replate the cro fragment.
Future Plan:
1. Promoter function verification
2. Plasmid construction of v40. Final with new cro plasmid
3. Serine integrase function verification: qPCR and microplate reading
Education and Communication
This week we plan to conduct a road show in schools, using games and posters to educate the college community about the importance of reproductive health and how to maintain their own reproductive health.Others
This week, we finished the promotion video! It was done very close to the deadline, everyone worked hard.
Summer is almost over, and the students who went home are coming back to school, so we had another potluck this weekend!
Week of August 29th to September 4th
Pmr and Amplifier
Week Plan:
1. Verification of LacZ-Toehold Switch 1&2 (If it still remains no result, we would give up Toehold Switch).2. Change the promoter of Toehold Switch-mCherry for a stronger one.
3. Verification of NisK/R-amplifier.
4. Construction of plasmid pBAD-T7T3.
5. Verification of amplifier system: T7-T3.
Progress:
1. Verification of LacZ-Toehold Switch 1&2 (If it still remains no result, we would give up Toehold Switch).The induction remained no result: no color change after being induced and added X-gal.
2. Change the promoter of Toehold Switch-mCherry for a stronger one.
We found a proper promoter, but Toehold Switch was given up.
3. Verification of NisK/R-amplifier.
We verified NisK/R-amplifier by EGFP fluorescence intensity using microplate reader.
4. Construction of plasmid pBAD-T7T3.
We successfully constructed this plasmid.
5. Verification of amplifier system: T7-T3.
We verified T7-T3 system (induced by pBAD) by EGFP fluorescence intensity using microplate reader.
Future Plan:
1. Verification of NisK/R-amplifier by FCM.2. Construction of plasmid J23100-AffiPmrBAC-T7T3-EGFP.
Nisin
Week Plan:
1. Use NisA-affi to induce expression.2. Change the expression strain to Mach1-T1
3. Construct the plasmid containing NisK with EGFP downstream
Progress:
1. Use NisA-affi to induce expression.The results looked better after changing the expression strain to Mach1-T1.
2. Change the expression strain to Mach1-T1
The Mach1-T1 strain had better performance in expressing EGFP and lacZ than BL21(DE3)
3. Construct the plasmid containing NisK with EGFP downstream(pcofocal)
We successfully construct the plasmid.
Future Plan:
1. Express pconfocol and detect the express of EGFP use fluorescence confocal microscope2. Use NisA-affi to induce expression: continue
3. Change the PnisA promoter according to software results, and conduct expression.
Logic Gate
Week Plan:
1. Promoter function verification2. Plasmid construction of v40. Final with new cro plasmid
3. Serine integrase function verification: qPCR and microplate reading
Progress:
1. Promoter function verificationResults from microplate reading indicated that the pBad promoter was functioning well without any leakage. Our induction condition (1% arabinose, 37℃) proved to be effective. The results also indicated that OR probably did not have effect of promoting transcription.
2. Plasmid construction of v40. Final with new cro plasmid
We replaced the cro fragment in the final plasmid. The sequencing result confirmed that our construction was successful.
3. Serine integrase function verification: qPCR and microplate reading
We transformed v41. Two in one plasmid into BL21(DE3) strain.
We inducted under the optimal condition (1% arabinose, 37℃) to test serine integrase function.
We performed qPCR after arabinose induction to test the inversion rate of serine integrase, yet no significant difference before and after induction can be seen.
We also measured the fluorescence intensity using microplate reader to examine the inversion rate of serine integrase.
Troubleshooting:
1. qPCR result:Problem:
No significant result can be seen for qPCR even though microplate reading indicated stark contrast of fluorescence intensity before and after the induction. Several wells even showed no ct after qPCR, yet correct bands can be seen after we ran the qPCR product on the gel.
Analysis:
We speculated that our qPCR system was somewhat inappropriate.
Solution:
We planned to optimize our qPCR protocol and system.
Future Plan:
1. Cro&cI function verification: test new cro
2. Serine integrase function verification: qPCR
Education and Communication
We finished planning the roadshow this week, but unfortunately the school rejected our application for the event as it is now in the event insurance period.September
Week of September 5th to 11th
Pmr and Amplifier
Week Plan:
1. Construction of plasmid J23100-AffiPmrBAC-T7T3-EGFP.2. Verification of AffiPmrBAC system by FCM.
Progress:
1. Construction of plasmid J23100-AffiPmrBAC-T7T3-EGFP.We successfully constructed the plasmid J23100-AffiPmrBA.
2. Verification of AffiPmrBAC system by FCM.
We verified the AffiPmrBAC system by FCM and the augmenter of EGFP fluorescence intensity indeed existed but was very low.
Future Plan:
1. Construction of plasmid J23100-AffiPmrBAC-T7T3-EGFP.2. Verification of NisK/R-amplifier by FCM.
Nisin
Week Plan:
1. Express pconfocol and detect the express of EGFP use fluorescence confocal microscope2. Use NisA-affi to induce expression: continue
3. Change the PnisA promoter according to software results, and conduct expression.
Progress:
1. Express pconfocol and detect the express of EGFP use fluorescence confocal microscopeThe result showed the transfer of NisK to cell membrane was normal, but there are large differences in different colonies.
2. Use NisA-affi to induce expression: continue
We had obtained a lot of data, some of which support our hypothesis.
3. Change the PnisA promoter according to software results, and conduct expression.
We successfully constructed the 10 plasmids(pRRH01-pRRH10)
Future Plan:
1. Induce pRRH01-pRRH10 and detect the expression of EGFP2. Use flow cytometry to detect the expression of EGFP
Logic Gate
Week Plan:
1. Cro&cI function verification: test new cro2. Serine integrase function verification: qPCR
Progress:
1. Cro&cI function verification: test new croWe transformed v40. Final with new cro plasmid into BL21(DE3) strain to test the function of cro and cI under arabinose induction.
We inducted under various conditions (1% arabinose concentration, different temperature, and time) to explore the optimal experiment condition for cro and cI to function.
We measured fluorescence intensity by microplate reader after arabinose induction to test the function of cro and cI.
2. Serine integrase function verification: qPCR
We repeated the experiment for several times only to receive similar results. Therefore, we consulted with other advisors and planned to optimize our qPCR protocol and system.
Troubleshooting:
1. Cro&cI function verification:Problem:
Under the optimal induction condition, cI and cro both seemed to function. However, cro seem to be less effective than the positive control.
Analysis:
Conclusions were too soon to be drawn before we repeated the experiment for several times.
Solution:
We planned to repeat this experiment.
2. qPCR result:
Problem:
No significant result can be seen for qPCR even though microplate reading indicated stark contrast of fluorescence intensity before and after the induction. Several wells even showed no ct after qPCR, yet correct bands can be seen after we ran the qPCR product on the gel.
Analysis:
We speculated that our qPCR system was somewhat inappropriate.
Solution:
We planned to optimize our qPCR protocol and system.
Future Plan:
1. Cro&cI function verification: microplate reading to test new cro
2. Serine integrase function verification: qPCR
Week of September 12th to 18th
Pmr and Amplifier
Week Plan:
1. Construction of plasmid J23100-AffiPmrBAC-T7T3-EGFP.2. Verification of NisK/R-amplifier by FCM.
3. Construction of plasmid pBAD24-EriaCFP.
Progress:
1. Construction of plasmid J23100-AffiPmrBAC-T7T3-EGFP.We successfully constructed this plasmid (tested by PCR), and the plasmid was sent for sequencing.
2. Verification of NisK/R-amplifier by FCM.
We verified the NisK/R-amplifier, the result showed that the NisK/R system didn't work well with the T7T3 amplifier system.
3. Construction of plasmid pBAD24-EriaCFP.
We successfully constructed this plasmid and expressed EriaCFP.
Future Plan:
1. Verification of AffiPmrBAC-amplifier system.2. Build a colourimetric card of EriaCFP and mCherry.
Nisin
Week Plan:
1. Induce pRRH01-pRRH10 and detect the expression of EGFP2. Use flow cytometry to detect the expression of EGFP
Progress:
1. Induce pRRH01-pRRH10 and detect the expression of EGFPThe results showed the new promoters were stronger than the original promoter, and the effect of induction improved at the same time.
2. Use flow cytometry to detect the expression of EGFP
The results are positive and the data is roughly consistent with our assumptions.
Week of September 19th to 25th
Pmr and Amplifier
Week Plan:
1. Verification of AffiPmrBAC-amplifier system.2. Construction of plasmid pET28a-AffiPmrB-aa.
1. Verification of AffiPmrBAC-amplifier system.
We verified the AffiPmrBAC-amplifier by EGFP fluorescence intensity using microplate reader, it showed a much better result than AffiPmrBAC after 10h induction.
2. Construction of plasmid pET28a-AffiPmrB-aa.
Future Plan:
1. Verification of AffiPmrB-aaAC.Nisin
Week Plan:
We have entered into a collaboration with HUST-China, and we will help them with the design of the experimental system. For this purpose, we need to help them to conduct induction experiments and observe the fluorescence by fluorescence microscopy.Progress:
This week, we completed an induction experiment with the bacteria provided to us by HUST-China. The bacteria were shaken to saturation overnight and inoculated into LB medium at a ratio of 1:50, and were induced by adding 0.5 mM IPTG and 50 μM Cu2+ at an OD of 0.6. After overnight induction, we centrifuged the organisms and washed them with PBS, and added primary and secondary antibodies for immunofluorescence reaction, and finally added 100 μL of PBS for filming and observation under fluorescence microscope.Troubleshooting:
In the results of this induction experiment, the number of fluorescent bacteria under the microscope was not very high, probably because the FITC-labeled secondary antibodies were not incubated strictly against light during the experiment. We will improve this in the next experiment.Future Plan:
We repeated the experiment and gave the results to HUST-China. we also discussed with them this week the results of the nisin induction experiment they performed for us.Week of September 26th to October 2nd
Pmr and Amplifier
Week Plan:
1. Verification of AffiPmrB-aaAC.Progress:
1. Verification of AffiPmrB-aaAC.Weerified the AffiPmrB-aaAC by EGFP fluorescence intensity using microplate reader, it showed a better result than AffiPmrBAC: a higher augmenter of EGFP fluorescence intensity and a better affinity with IgG.