2022-06-01~2022-06-07  

Select the team members for the experiment, wiki, human practice, modeling, and artwork respectively and form a team of 15 members.
We analyzed iGEM competition and shared our understanding of it collectively.
After communicating with our PI, we finally selected our topic this year----Chlipid: a highly productive biofuel factory.

2022-06-08~2022-06-14  

Our team members looked up and read the literature extensively, and finally settled the culture, transformation, gene editing, and gene expression methods of Chlamydomonas reinhardtii, as well as the according design and improvement of the Crispr/cas9 system.
Our team members did cas9 plasmid and sequence summary, gene transformation system construction, lipid gene and the summary of protocols.

2022-06-15~2022-06-30  

We Started to experiment with different culture methods, metabolic pathway studies and CRISPR system selection for Chlamydomonas reinhardtii.
We designed 6 Composite Parts, they are HSP70A Promoter+3×Flag+NLS+SpCas9+NLS+Rbcs2 Term、U6 promotor+insert site+SpScaffold+polyT Term、Rbcs2 Promotor+APHVII+Rbcs2 Term、Rbcs2 Promotor+mCherry+Rbcs2 Term、Rbcs2 Promotor+Staygold+Rbcs2 Term.
We sent the sequences we needed to Tsingke Biotechnology Co., Ltd for synthesis, and got our synthesized sequence fragments at the end of June.

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2022-07-01~2022-07-07  

We designed and improved the following experiments: Chlamydomonas reinhardtii culture experiment, paddle plate conservation and conservation recovery amplification experiment, TEA electrophoresis experiment, electro-transformation and glass bead transformation experiment, OD measurement by Thermo Fisher ELISA, and plasmid extraction protocol.
We reviewed the literature to identify genes and pathways in the metabolic pathway of Chlamydomonas reinhardtii that can significantly affect its lipid production.
We did liquid and solid cultures of Chlamydomonas reinhardtii and finalized six different stress conditions to be performed on Chlamydomonas reinhardtii: N stress, Fe stress, salt stress, N+S stress, N+P stress, and S+P stress.
We found that our cultivation of Chlamydomonas reinhardtii was contaminated by stray bacteria, so we disinfected our laboratory and light incubator.

2022-07-08~2022-07-14  

We have designed and improved the protocol for lipid extraction.
We reviewed the literature and identified seven genes to be knocked out by the Crispr-cas9 system to promote lipid production in Chlamydomonas reinhardtii: LACS1, LACS2, DTH1, CHT7, PEPC1, ACX2, PLA2, and ICL1.
We designed protocols for six stress conditions of Chlamydomonas reinhardtii.
Under the guidance of our advisor Xu Tang we started to build the CRISPR-Cas9 system plasmid.

2022-07-15~2022-07-21  

We designed experiments to measure the OD values and cell numbers at 750 nm of Chlamydomonas reinhardtii at different gradient dilutions under normal culture conditions, and worked with modeling members to establish the growth curve of Chlamydomonas reinhardtii under normal cultivation.
We obtained the plasmid PZHY989 (with fluorescent protein YFP), which was kindly donated by the university laboratory, and used it to start experimenting with glass bead transformation and electrotransformation methods, while performing a series of experiments exploring concentration gradient screening pressure, glass bead size, and electrotransformation parameters.
We tried enzymatic digestion and gel recovery experiments. After running the gel, we found that the striped loop plasmid was not cut by the enzyme and the concentration of the plasmid after gel recovery was too low, so we looked up the literature and designed different experiments, trying to solve the problem, but the result is rather than satisfactory.

2022-07-22~2022-07-31   

We continued the experiments of enzyme digestion and gel recovery, made continuous improvement to the protocol, and finalized our solution of enzyme digestion overnight and gel recovery in the absence of gel. Finally, we obtained good results.
We Started experimenting with Nile Red staining and kept improving the protocol while observing the staining results under fluorescence microscope.
We did pre-experiments with six groups of stress conditions. Besides, a method was devised for freezing and preserving the seeds of Chlamydomonas reinhardtii.
We improved the treatment conditions in the electrotransformation protocol we learned from others, made a second attempt, and set up different concentration gradients of screening pressure for thaumatin so as to find the most suitable conditions for selecting algae.
We constructed the plamid which involves CRISPR-Cas9 system successfully and started to design sgRNAs.

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2022-08-01~2022-08-07   

We found false positives of our algae after electrotransformation. After communicating with our PI, we decided to use a 96-well plate for the secondary selection of our algae.
Based on the results of the plate after the second electrotransformation, we determined the screening pressure of 25 μg/mL as the screening pressure after electrotransformation, which laid the foundation for the later electrotransformation experiments.
The modified method of plasmid mini extraction reduces the loss of reagents.
The most suitable Nile Red staining method was explored from the variables of microwave heating time, whether DMSO was added or not, and the staining solvent.
We finished the design of all the sgRNAs and sent them to the company for synthesis and started to insert our sgRNAs into the insert site of our system.

2022-08-08~2022-08-14   

We summarized the experience of electrotransformation and designed a protocol for secondary screening and subsequent observation of fluorescence based on the grown single algal colonies.
Due to the low conversion rate and a low number of positive clones in the early stage resulted from electrotransformation, we retrieved the factors which influence the electrotransformation efficiency and designed two more sets of electrotransformation protocols which were remained to be tried.
We did the in-depth drawing of the growth curve for algae of WT and algae under Fe and N stress conditions, Nile red staining, and quantitative analysis of fluorescent images experiments.
We did the first round of fragment insertion. And we found that only a few succeeded, and the control groups of DH5α transformation were often contaminated with spurious bacteria.

2022-08-15~2022-08-21   

Based on the experimental results, we decided to change the antibiotic used for antibacterial purposes from Amp to Tim. The concentration of usage was 250 μg/mL. These gestures led to the reduction of the miscellaneous bacteria.
In our constructed plasmid with empty vectors (pTX2038, pTX2039, etc.) as well as plasmid vectors that have been combined with different gRNAs, different electrotransformation protocols were tried while performing formal electrotransformation experiments with these materials in the hope of obtaining optimal processing conditions.
We learned to use a fluorescence spectrophotometer and performed quantitative analysis of Nile Red staining for a new set of stresses.
We consulted our PI and Advisor for the advice on the second round of fragment insertion, after which process we increased the cycle number of Golden Gate assembly. Although we focused on an aseptic operation to avoid contamination by spurious bacteria, the insertion success rate was still not high.

2022-08-22~2022-08-31   

We purchased the Electroporation Buffer (ME-Suc): MAX Efficiency™ Transformation Reagent (therfisher science, #A24229). Subsequently, the electrotransformation experiments were performed according to its instructions. And the positive results were obtained, so we decided to continue using this protocol for subsequent electrotransformation experiments.
We did a quantification of Nile Red staining results by using a fluorophotometer.

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2022-09-01~2022-09-07   

Positive clones were obtained by electrotransformation and target bands were successfully verified by algal colony PCR.
In the following stage, we continuously did electrotransformation experiments.
The glass bead transformation experiment was continued and single algal colonies were found to grow in the blank group during the glass bead process.
We did the third round of plasmid construction, this time we were more careful. Finally our success rate was much higher though certain failure still exists.

2022-09-08~2022-09-14   

We continuously did electrotransformation experiments.
The glass bead conversion experiments were continued and the screening pressure of glass bead conversion was re-executed.

2022-09-15~2022-09-21   

Continuously doing electrotransformation experiments

2022-09-22~2022-09-30   

Continuously doing electrotransformation experiments.

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2022-10-01~2022-10-07   

We organized the data and results of the stress conditions.
We selected appropriate pictures for our various experiments.
We organized the results of transcriptome analysis and then wrote our wikipage.

2022-10-08~2022-10-12   

We organized and analyzed our previous results, and then wrote our wiki.

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