Team:FZU-China/Calendar

Notebook

3.18-3.22

We held the first meeting to discuss the direction of our project. After the meeting, PI asked everyone to check the feasibility of their ideas and sort the information. We sent documents to PI for review.

3.24

After the members’ reports, our PI decided to adopt Shanyu Huang’s project： using optogenetic switches to treat depression. And confirmed the final blueprint of the project: We hope that one day in the future when patients are basking in the sun, the special cell in their body will produce antidepressant substances to treat depression. PI asked everyone to check the information to confirm the project framework. The division of tasks was as follows: Xinyue Zhuang, Sipeng Chen, and Shiyang Jiang look up optogenetic switches; Huimin Zhang, Shanyu Huang, Ziqing Liu, and Jiale Hong look up antidepressant substances.

3.25

The queried proteins related to optogenetic mechanisms: ChR2, NpHR, ChIEF, VChR1. Inquired substances related to the antidepressant mechanism: serotonin, epinephrine, tyrosine, dopamine, folic acid, serotonin, endorphins, GABA, BDNF.

3.28-3.29

Exchange progress among undergraduates. We found that: 1. VTA pathway, PI3K-Akt pathway, interferon-α-induced glucocorticoid, and serotonin receptor regulation play a huge role in anti-depression; 2. Vascular endothelial growth factors and intestinal flora play a huge role in depression 3. UVR-8, AsLOV2, MAPK pathway, and VVD appear in many scholars' papers on optogenetics; 4. ChR2 double mutants (Asp156Ala and Cys128Ser)-SSFO, a new type of opsin have stronger photosensitivity, depolarization faster; 5. The connection between serotonin and the blood-brain barrier; 6. AsLOV2, VVD, FKF1, GI, UVR8, Cry2, CIB1, CPH1, PhyB, PIF, BphP1, BphP1 and other proteins The mechanism of action and wavelength value.

3.31

PI pointed out that the practicability of partial neurological schemes is lower than that of partial synthetic biology schemes. Our PI also recommended starting from the source of depression like mTOR pathway. After discussions, PI confirmed that the information to be checked next will focus on three parts: 1. What activates the photoprotein; 2. What protein should be set as the key substance to make the antidepressant effect; 3. How to use negative feedback regulation. It is worth noting that during the discussion, many undergraduate members proposed that a luminous substance (Equilin) could be found in the luminous jellyfish to activate the subsequent mechanism.

4.1

After discussions, our team added to our blueprint: eating a certain food, a certain ingredient in this food activates our synthetic biology system. It is preliminarily determined that module one focuses on the subsequent molecular mechanism of Aequorin's blue light activation; the two directions of the module are preliminarily determined as mTOR pathway and PI3K-Akt pathway, and the antidepressant effect is exerted by regulating the expression of a certain substance on this route; preliminarily determining the module Third, a negative feedback system will be established to regulate antidepressant substances.

4.3

Undergraduates communicate on progress. At the same time, real-time search and discussion are carried out to ensure that the biological components used in the entire synthetic biology system are confirmed as soon as possible. We inquired: 1. Ketamine has a rapid antidepressant signal pathway; 2. Equiline's mechanism ; 3. Depression is often accompanied by inflammation and is related to the inhibition of the GR pathway; 4. Jellyfish luminescence The luminescence reaction of protein is irreversible. Only by removing calcium ions and adding reduced jellyfish fluorescein can aequorin can receive calcium ions to trigger the luminescence reaction; 5. The method of introducing Equiline into cells and measuring intracellular Calcium ion method; 6. Basic knowledge of growth factors, Akt/PKB, PDK2, Rac-1, Cdc42 and other substances involved in the mTOR pathway; 7. Serotonin mechanism of action. At the same time, during the review process, we realized that the use of aequorin may be difficult to carry out experiments, one is because the difficulty of synthesis is unknown, and the other is that the finished product is expensive.

4.5-4.7

We found that BDNF, GABA and insulin growth factor (IGF) are closely related to the antidepressant effect. At the same time, it was confirmed that ketamine and serotonin are not suitable as the key substances of this project. The first reason is that it is a controlled drug, and the other is The mechanism of the blood-brain barrier is complicated. We also looked up the gene sequence of Aquilin protein and its relationship with coelenterazine, as well as the improvement of its expression in Escherichia coli. At the same time, we found two photoprotein genes, aeqxm and aeqxxm, similar to Aquelin-derived genes. But in the end, given that aequorin is more difficult to operate and implement related mechanisms, we decided to abandon aequorin and decide to find a suitable structural photoprotein.

4.8

Hold a lunch meeting. After the discussion, module one was preliminarily confirmed: pLac-aeqxm and pTet-aeqxxm emit light with a wavelength of 470nm under the action of calcium ions to activate the optogenetic switch. Module 2: Overexpression of IGF to exert an antidepressant effect. Module 3: Set up a bacterial self-detonation mechanism so that IGF can leave the engineered bacteria and enter the human body to play a role. But afterward, we found that the document with the view that "IGF is a suitable antidepressant" refers to IGF as a small molecule terpenoid, not a protein. Therefore, we continue to divide tasks to find suitable biological components.

4.11-18

We have checked: 1. Appropriate components of the self-detonation mechanism-φX174E and E.coli E7; 2. The previous researcher's bacterial lysis model in E. coli centered on φX174E; 3. VEGF and FGF2 expression and interaction The role is related to the antidepressant mechanism, but due to the complexity of the mechanism, it has not been adopted; 4. Glutamate receptors play an important role in the antidepressant mechanism, which is related to the previously queried GABA; 5. LacZ, Physiological effects of LacI, LacO and other substances; 6. Many studies on optogenetic switches by Jeffrey J. Tabor and Michael B. Sheets. The special biochemical process of Cre and VVD makes them enter the candidate range; 7. IPTG was accidentally checked; 8. It was checked that the increase in BDNF content would enhance the antidepressant response, and BDNF entered the candidate range.

4.20

Progress discussion among members. After discussion, four modules are finally formed: module one is a light-emitting module; module two is an optogenetic switch module; module three is an antidepressant module; module four is a cracking module. However, we found that the wavelength of light emitted by the photoprotein selected in the currently confirmed optogenetic system may not be suitable, so we need to find a new photoprotein.

4.21-4.27

We found: 1. The Cre-loxP recombinase system further established the inevitability of the selection of Cre and VVD; 2. VEGF also plays an antidepressant effect, but this conflicts with some literature. After a long period of searching information and considering the source of antidepressant substances, as well as the idea that "eating certain foods can activate antidepressant substances" long ago, it was finally decided that GABA was the key substance of our project.

4.28

We confirm the use of gadB to catalyze MSG to increase the GABA content. And confirm that the preferred solution for the cracking module is φX174E. At the same time, in consideration of the activity of the activating protein, the PI suggested replacing the biological components in the first module.

4.29

The PI suggested that if we want to use aequorin in intestinal bacteria, we must also consider how to activate aequorin. This problem has become our concern. Module 1 must be replaced with new biological components.

4.30-5.14

We found that Luciferase is more suitable for the system we currently build, and there is no need to add any additional small molecules. It was confirmed that the photoprotein was replaced with LuxCDABE, and the project framework was settled. PI Shaobin Guo, graduate student PI Jingjing Lin, and team leader Huimin Zhang discussed the preparation and source of each biological component and started to order some components. Members began to learn how to use websites such as UniProt and Addgene to search for the structure and nucleic acid sequence of each biological element. Members formally entered the laboratory to learn about gel making, electrophoresis, PCR equipment and other related operations. Jingjing Lin guides the undergraduate members to learn to use snapgene to design protein sequence primers. The relevant primer design is completed. Learn Snapgene plasmid ligation operation.

5.31-6.06

Undergraduate members enter the laboratory to learn basic operations such as the use of centrifuges, LB medium preparation, and PCR product recovery.

6.07-6.13

Module 3: In order to realize the production of GABA, we determined the gene sequence of the gadB protein that can interact with the substrate L-glutamic acid, designed the gadB-BsaⅠ bidirectional primer with the BsaⅠ restriction site, and obtained it from BL21(DE3) by PCR. ) To obtain the gene sequence of gadB-BsaI.

Module 4: In order to achieve the final lysis of the bacteria in the project, we found the gene company to synthesize the lysis gene φX174E-BsaⅠ (in order to maintain its stability, the company cloned it into pUC57). In order to achieve the unification of the plasmid backbone in the subsequent experiments, we re-purified the product after PCR and demethylation digestion to obtain φX174E-BsaⅠ linear DNA, and performed PCR and demethylation digestion on the plasmid pSG86 in the laboratory. The product was purified to obtain the plasmid backbone v1-1-pT7-RBS-T500(2)-BsaI.

6.14-6.20

Module 3: Connect the v1-1-pT7-RBS-T500(2)-BsaⅠ and gadB-BsaⅠ obtained last week to GGA. After the connection is completed, perform the chemical transformation of the ligation product. Check whether there is a single colony on the plate. Preliminarily judge whether the connection is successful. The chemically competent cell we selected was the cloning bacterium Top10. After overnight culture at 37°C, a single colony appeared on the plate. Colony PCR was performed. Through nucleic acid gel electrophoresis, we saw a bright band at 1666bp. After the bacteria, we send the constructed plasmids to the gene company for sequencing and the bacteria preservation.

Module 4: Connect the v1-1-pT7-RBS-T500(2)-BsaⅠ, φX174E-BsaⅠ obtained last week to GGA connection. After the connection is completed, the chemical conversion of the connection product will be carried out. The experiment process is the same as that of Module 3. A single colony on the plate grew normally, but after performing PCR and nucleic acid electrophoresis, the bands obtained were incorrect. The reason may be that the plasmid backbone has been self-linked and not connected to φX174E-BsaI.

6.21 to 7.11

Preparing for the final exam

7.12-7.18

Module 2: In order to realize the regulation of gadB protein expression in the project, we found a gene company to synthesize loxP-TT-loxP-BsaⅠ (in order to maintain its stability, the company cloned it into pUC57), and obtained loxP-TT- by PCR LoxP-BsaⅠ linear DNA, demethylation digestion, purification of the product, and GGA ligation, through the transformation overnight culture, there is no colony on the plate, indicating that the connection failed, re-attach.

Module 3: Extract the constructed plasmid v1-1-T7-RBS-gadB-T500(2) from the cloned bacteria, and transform the plasmid into BL21(DE3). After culturing overnight at 37°C, place it on the plate A small number of small single colonies appeared, and nucleic acid electrophoresis was performed after colony PCR, and there was no band. The reason may be contamination by bacteria.

Module 4: Since the v1-1-pT7-T7RBS-sfGFP-T7RBS-φX174E-T500(2) plasmid was not successfully constructed last week, the ligation product was re-ligated through GGA and transformed into TOP10 for overnight culture at 37°C. A single colony on the plate grew normally, but the band of the electrophoresis result after PCR of the bacterial solution was incorrect. After reflection, it was found that the reason was the addition of the wrong ligation product.

7.19-7.25

Module 1: Plasmid extraction of pW34-LuxCDABE (Apr) from the Pir1 strain purchased from addgene, because the subject wants to achieve the co-transformation of the three plasmids, in order to prepare in advance, transform pW34-LuxCDABE (Apr) into BL21 (DE3) In ), after overnight culture at 37°C, the plate did not grow bacteria. Analysis of the reason: it may be that the plasmid is too large to be 8kb, and it is more difficult to transfer into BL21(DE3) bacteria.

Module 2: Plasmid extraction from pBbS5a-Cre-VVD (Amp) from the DH5α strain purchased from addgene, because the subject wants to achieve the co-transformation of the three plasmids, in order to prepare in advance, transform pBbS5a-Cre-VVD (Amp) To BL21 (DE3).

Module 3: The plasmid v1-1-pT7-RBS-gadB-T500(2) was not successfully transformed into BL21(DE3) last week, so this week is mainly to obtain v1-1-pT7-RBS-gadB-T500( 2) Plasmid BL21 (DE3), through re-transformation, there are more single colonies growing on the plate. Pick 4 single colonies and perform PCR with the bacterial solution. The gel running results are good. Select bacteria No. 2 for overnight shaking at 37°C. The strain was preserved in two days.

Module 4: Reconstruct v1-1-pT7-T7RBS-sfGFP-T7RBS-φX174E-T500(2) through GGA connection, and transform it into TOP10 overnight culture at 37°C, a single colony appears on the plate the next day, pick it Four single colonies were subjected to bacterial liquid PCR, and the gel running results were good. The bacteria No. 3 and 4 were selected for overnight shaking at 37°C, and the next day they were sent for sequencing and culture preservation.

7.26-8.1

Module 1: Since no single colony was obtained from chemical transformation, the reason for analysis may be that the plasmid is too large and the transformation efficiency is low. So electroporation was used to transform pW34-LuxCDABE (Apr) into BL21 (DE3) and cultured overnight at 37°C Later, the plate grew bacteria, but there was no band in the bacterial liquid PCR gel. Reason: The replication initiation site of this plasmid is special and requires special strains to be preserved. Later, luxCDABE is connected with other plasmid backbones.

Module 2: Since we forgot to add the gene element of T7 promoter when constructing V1-1-pT7-loxP-TT-loxP-T7RBS-sfGFP-T500(2) plasmid in the early stage, so the V1-1-loxP-TT-loxP -T7RBS-sfGFP-T500(2) Plasmid was subjected to PCR to obtain V1-1-loxP-TT-loxP-T7RBS-sfGFP-T500(2)-BsaⅠ linear DNA with BsaⅠ restriction site, and then proceed with pT7 GGA connection, colony selection for bacterial liquid PCR, through nucleic acid electrophoresis experiment, there is a bright band at 1172b, and the remaining bacterial liquid is shaken overnight at 37°C and sent to the company for sequencing the next day.

Module 3: Query-related experimental conditions and basic principles of gadB catalytic glutamate. A small amount of BL21(DE3) containing v1-1-pT7-RBS-gadB-T500(2) plasmid was deducted from -80℃ and cultured overnight. The next day, 1% bacterial solution was added to 100mL of fresh medium Carry out expansion training (37°C, 250rpm). Measure OD600, add IPTG to induce overnight (16°C) when OD600 is 0.6 to 0.8. Then the cells were collected by centrifugation, and the supernatant and precipitate were obtained by centrifugation after high-pressure sterilization. The supernatant was purified using imidazole gradients of different concentrations. Aspirate 20 μL of different eluted samples and perform SDS-Page protein gel electrophoresis. After the protein gel is obtained, it is stained with Coomassie Brilliant Blue, and a single band is obtained after decolorization. However, the band is less obvious, probably because the product yield is low. Expand the bacterial solution to 400 mL, and redo it the next day.

Module 4: The sequencing result is not ideal, the base mutation occurs, the stop codon appears in φX174E, and the plasmid cannot be used. Re-connect with GGA to construct v1-1-pT7-T7RBS-sfGFP-T7RBS-φX174E-T500(2) plasmid.

8.2-8.8

Module 2: V1-1-loxP-TT-loxP-T7RBS-sfGFP-T500(2) Plasmid sequence is correct, the extracted plasmid is pBbS5a-Cre-VVd, v1-1-pT7-loxP-TT-loxP-sfGFP-T500( 2) Preparation for co-transformation of double plasmids.

Module 3: Learn the principles and basic operations of high performance liquid chromatography, check the literature to confirm the method of using HPLC to determine GABA, and write an experimental plan for using HPLC to determine GABA standards.

Module 4: Transform the reconstituted plasmid v1-1-pT7-T7RBS-sfGFP-T7RBS-φX174E-T500(2) into competent TOP10, and cultivate overnight at 37°C. The colonies on the plate grew normally. Perform colony PCR and found a bright band at 1267bp. The plasmid v1-1-pT7-T7RBS-sfGFP-T7RBS-φX174E-T500(2) was extracted the next day, and the sequencing results were good. The plasmid was transformed into BL21(DE3).

FZU-China for FAFU-China: After receiving the b3 plasmid sent, transfer the b3 plasmid into BL21(DE3). Pick 6 single colonies for bacterial liquid PCR, electrophoresis to run the gel, the results show that the band is correct. Divide the bacterial solution into two groups with three tubes in each group, and then expand the culture (50 mL) after small culture, and then culture the first group in a blue light environment and culture the second group in a dark environment. Finally, under the same OD600, 1μL of the bacterial solution was sucked on the agar plate, and it was found that more green fluorescent bacteria can be seen under blue light conditions, but only a small amount of green fluorescent bacteria under dark conditions.

8.9-8.15:

Module 2: Extract pBbS5a-Cre-VVd, v1-1-pT7-loxP-TT-loxP-sfGFP-T500 (2) high-concentration plasmids, and use them to express in the TXTL system to verify whether Cre recombinase can be expressed in vitro Identify the loxP site and cut it.

The pBbS5a-Cre-VVd, v1-1-pT7-loxP-TT-loxP-sfGFP-T500(2) double plasmids were co-transferred to the electro-competent BL21 (DE3) for subsequent in vivo Opto-Cre-Vvd double Validation of plasmid system. Pass ①②group whether to give light to the two experimental groups and ③Pos.Control (BL21(DE3) containing v1-1-pT7-RBS-sfGFP-T500(2)) ④Neg.Control (without plasmid) ⑤Contain v1-1 -pT7-loxP-TT-loxP-sfGFP-T500(2) single plasmid BL21(DE3), a total of 5 sets of experiments, the results found that after adding IPTG, ①②③⑤ all have green fluorescence, initially suspected to be BL21(DE3) competent The quality of the bacteria caused the background leakage, so all BL21(DE3) were replaced with the BL21(DE3) competence of Tiangen Company, and then it was found that there would still be serious leakage. Check the plasmid backbone of v1-1-BsaⅠ It was found that the lack of two regulatory elements, lacO and lacI, on the backbone resulted in serious leakage of the plasmid. Therefore, it was decided to replace all the plasmid backbones of v1-1-BsaI with pET30a-BsaI.

Module 3: Carry out HPLC experiments. It is divided into two parts: standard curve determination and sample determination. Standard curve determination part: configure the sodium bicarbonate solution, phosphate buffer, FDNB solution, GABA standard gradient concentration solution required for derivatization. Prepare the mobile phase required for the experiment, and then perform suction filtration and ultrasonic defoaming on the mobile phase. After derivatizing the gradient concentration solution of the GABA standard substance according to the steps, it is suction filtered, and it is kept in a dark ice bath for later use. After the baseline is stable, the derivatized standard curve solution is measured and the standard curve is drawn. Sample measurement part: and adding the substrate L-glutamic acid to the gadB enzyme solution, after the substrate reaction, derivatize the blank reagent, GABA standard and the enzymatic reaction solution, and use HPLC to measure the sample, but halfway through The HPLC pressure line is unstable, so this experiment lacks credibility.

Module 4: As the optogenetic switch part needs to replace the skeleton with pET30a, in order to ensure the consistency of subsequent experiments, the cleavage module is also replaced with the same skeleton. The previously transformed plasmid cannot be used and the plasmid needs to be rebuilt. Re-obtain the backbone pET30a-BsaI and the cleavage gene ΦX174E-BasI, and connect them with GGA, connect the backbone pET30a and the cleavage gene ΦX174E through GGA and chemically transform to obtain the plasmid pET30a-ΦX174E.

8.16-8.22

Module 2: GGA is connected to pET30a-BsaⅠ, pT7-loxP-TT-loxP-sfGFP-BsaⅠ and the connection product pET30a-pT7-loxP-TT-loxP-sfGFP is transformed into DH5α. After overnight culture at 37℃, the successful cultured single The colony was subjected to colony PCR, and a bright band at 1438 bp was obtained by nucleic acid gel electrophoresis, indicating that the ligation product in this single colony was correct. After shaking the bacteria overnight, the strains are preserved and the plasmids are extracted and sent to the gene company for sequencing. Sequencing results showed that there was no problem with the plasmid. The pET30a-pT7-loxP-T-T-loxP-sfGFP (Kana) was transformed into BL21 (DE3) and the strain was preserved.

Module 3: Small training and expansion of BL21(DE3) containing v1-1-pT7-RBS-gadB-T500(2) plasmid, and induce the expression of gadb protein, perform protein purification and replace the buffer of gadb protein, remove the original buffer The imidazole, and the concentrated protein solution.

Module 4: Take out the bacterial solution after overnight culture, extract the plasmid, and send it to the company for sequencing. The sequencing results showed that the plasmid was no problem. The extracted plasmid was transferred into BL21(DE3) competent cells, subcultured and then expanded (50mL), and the OD600 value of the bacterial solution was measured at intervals (picture). When OD600=0.6, 50mL of the bacterial solution was divided into two groups. After 3.5 hours, IPTG was added to one bottle of bacterial solution (to make the final concentration 100μM) for induction, and the other bottle was not added with IPTG. It can be observed that the OD value of the IPTG group continues to decrease while the OD value of the non-IPTG group continues to increase, and it can be clearly observed that the bacterial solution of the IPTG group has become clear, and the bacterial solution of the non-IPTG group is still turbid ( Picture), which shows that the bacteria added to the IPTG group are lysed. The cracking part is over.

8.23-8.29

Module 2: Co-transform the pET30a-loxP-TT-loxP-sfGFP (Kana) plasmid and pBbS5a-Cre-VVd (Amp) with two plasmids, culture overnight at 37°C, the colonies on the plate grow normally, and the colonies pass through the nucleic acid gel after PCR Obvious double bands appeared in electrophoresis, proving that the double plasmid co-transformation was successful.

Module 3: Using Berthelot's method to determine whether GABA is produced or not, the absorbance is measured at 630nm. Reaction steps: prepare a phenol solution with a mass concentration of 6%, 5 mL of 200 mM sodium borate, and a 5% sodium hypochlorite solution. Dilute the 2 mg/mL GABA solution to 1 mg/mL, 0.8 mg/mL, 0.6 mg/mL, 0.4 mg/mL, 0.2 mg/mL and ice bath with the blank reagent. Add 500 μL of water, 500 μL of 6% (v/v) phenol solution, 100 μL of 200 mM sodium borate (pH 9.0), and 400 μL of 5% (w/v) sodium hypochlorite solution into six test tubes. , And then add 1 mL of GABA solution of different concentrations and 1 mL of water in the blank control. Put the test tube in a water bath at 100°C for 10 minutes, take it out and place it on ice for 20 minutes. Measure the absorbance at 630nm. It was found that the blue-green reaction solution was not obvious, and there was no difference in the absorbance at 630nm of each group of different concentrations of GABA measured under the UV spectrophotometer. The reason is that the concentration gradient is set too close, and the detection limit may not be as expected. high.

8.30-9.5

Module 3: The HPLC determination of the GadB enzymatic reaction product is as follows: Enzymatic reaction: construct a 2.0mL reaction mixture system: 1. Prepare Na2HPO4 solution (7.71mL 0.2M), mix with citric acid (12.29mL 0.1M) to obtain 200mM Na2PHO4-citric acid buffer (pH4.0), 50mM L-gluten Acid, 0.01mM PLP, 50-100 μL purified enzyme. Fully mix the above system, incubate at 37°C for 2 hours, take the reaction solution every 30 minutes during the incubation process, boil it for 5 minutes and inactivate it, put it in a refrigerator at 4°C after labeling, and proceed immediately to the derivatization reaction. Derivatization: Dissolve 1% FDNB with acetonitrile, and then store it at 4°C in a dark environment. Prepare eight empty test tubes and mark them as blank, GABA standard solution, 0.5h, 1h, 1.5h, 2h, 0.5h+GABA, 1.5h+GABA. Preparation system: 250μL boiled and inactivated sample + 250μL sodium bicarbonate + 250μL 1% FDNB solution Note: The sample added to the test tube marked as blank is phosphoric acid-citric acid buffer solution+L-MSG+PLP+FDNB+sodium bicarbonate. GABA was weighed to obtain 0.0291g, divided into three evenly, each of which was 0.0097g. One tube of GABA standard is dissolved in water and the volume is adjusted to 250μL. The test tubes labeled 0.5h+GABA and 1.5h+GABA required GABA to be dissolved in the reaction solution. Finally, all test tubes were made up to 2.5 mL with PBS solution.

HPLC experiment: The mobile phase of pump A is 50% acetonitrile; the mobile phase of pump B is PBS, and each injection is 20μL. Sample: blank, GABA standard, 1.5h, 1.5h+GABA. The pressure of pumps A and B is about 11.6MPa. The single operation cycle is 30min. Experimental result: Because I ran the derivatized enzyme solution with HPLC before, it was found that the peak time of the derivatization reagent shifted forward, so this experiment chose to add standard GABA after the enzymatic reaction 0.5h and 1.5h. Whether the peak time of GABA is consistent or not, the experimental results show that the peak time of the derivatization reagent still shifts forward. It may be that there are other substances in the enzyme solution that can react with FDNB before GABA. No solution has been found yet.

9.06-9.12

Module 1: GGA connects pACYCDuet-1-BsaⅠ, pre-luxCDABE-BsaⅠ, post-luxCDABE-BsaⅠ and transforms the connection product pACYCDuet-1-LuxCDABE to DH5α. After overnight culture at 37℃, colony PCR is performed on the successfully cultured single colony, Nucleic acid gel electrophoresis showed a bright band at 6641bp, indicating that the ligation product in this single colony was correct. After shaking the bacteria overnight, the strains are preserved and the plasmids are extracted and sent to the gene company for sequencing. Sequencing results showed that there was no problem with the plasmid. The pACYCDuet-1-LuxCDABE (Chl) was transformed into BL21 (DE3) and the strain was preserved.

Module 3: Expand BL21 (DE3) containing v1-1-pT7-RBS-gadB-T500(2) plasmid, use 400 mL of LB medium, 400 μL of antibiotics, about 4 mL of bacterial solution, and culture at 37°C on a shaker at 250 rpm to obtain gadB enzyme solution. Measure the absorbance of GABA at 485nm.

Derivatization is carried out in accordance with the FDNB method. GABA standard solution gradient: 0.5mg/mL, 1.0mg/mL, 1.5mg/mL, 2.0mg/mL, 2.5mg/mL, 3.0mg/mL blank control was replaced with the same amount of water. The measured absorbance at 485nm is as follows:
Blank control: -0.02
0.5mg/mL: 0.31
1.0mg/mL: 0.25
1.5mg/mL: 0.33
2.0mg/mL: 0.36
2.5mg/mL: 0.31
3.0mg/mL: 0.30

The result is not ideal and needs to be tried again.

9.13-9.26

The members of the experimental group are unfortunately in quarantine due to COVID-19 cases resurgence in our province.

9.27-10.2

Module 2: In vivo verification of Opto-Cre-Vvd double plasmid pET30a-loxP-T-T-loxP-sfGFP (Kana) & pBbS5a-Cre-VVd (Amp) system
①Opto-Cre-Vvd [pET30a-loxP-T-T-loxP-sfGFP & pBbS5a-Cre-VVd] +IPTG+Light
②Opto-Cre-Vvd [pET30a-loxP-T-T-loxP-sfGFP & pBbS5a-Cre-VVd] -IPTG+Light
③Opto-Cre-Vvd [pET30a-loxP-T-T-loxP-sfGFP & pBbS5a-Cre-VVd] +IPTG-Light
④Opto-Cre-Vvd [pET30a-loxP-T-T-loxP-sfGFP & pBbS5a-Cre-VVd] -IPTG-Light
⑤Pos.Control [pET30a-sfGFP] +IPTG+Light
⑥pET30a-pT7-loxP-T-T-loxP-sfGFP+IPTG+Light
⑦Neg.Control [BL21(DE3) without plasmid] +IPTG+Light

The results showed that the fluorescence value of group ②④⑥ was close to that of ⑦Neg.Control, but the fluorescence value of group ①③ with IPTG was close to that of ⑤Pos.Control. It is believed that the possible reason is that it is not strictly protected from light or the Cre enzyme has leaked expression.

Module 3: The results of the last experiment were not very satisfactory. GABA was derivatized with FDNB and then measured by colorimetry. The obtained standard curve and color proved that the method is feasible. The result of the gadB enzymatic reaction showed that the UV absorption value at 485nm was about 1.16, which proved that GABA was indeed produced.

10.3-10.10

Module 2: According to the recommendations of the NUDT instructor, the three groups of ①Opto-Cre-Vvd+IPTG+Light ③Opto-Cre-Vvd+IPTG-Light ⑥pET30a-pT7-loxP-TT-loxP-sfGFP were sampled for colony PCR. The result ① is clear at 913bp The band of, proves that Cre has been cut; ③⑥There is a clear band at 1113bp indicating that it has not been cut, indicating that the Cre enzyme is not completely leaked. Improve the experimental plan to strengthen the protection from light, and continue to verify the problem of Cre background reorganization and leakage. In vivo verification of Opto-Cre-Vvd double plasmid pET30a-loxP-T-T-loxP-sfGFP (Kana) & pBbS5a-Cre-VVd (Amp) system
①Opto-Cre-Vvd [pET30a-loxP-T-T-loxP-sfGFP & pBbS5a-Cre-VVd] +IPTG+Light
②Opto-Cre-Vvd [pET30a-loxP-T-T-loxP-sfGFP & pBbS5a-Cre-VVd] +IPTG-Light
③Pos.Control [pET30a-sfGFP] +IPTG+Light
④pET30a-pT7-loxP-T-T-loxP-sfGFP+IPTG+Light
⑤Neg.Control [BL21(DE3) without plasmid] +IPTG+Light

It was found that the fluorescence value of group ③ was close to that of ⑦Neg.Control, and the fluorescence value of group ① was close to that of ⑤Pos.Control, but the end-point fluorescence value of ② was about half of that of ①, indicating that there was a small amount of background leakage of Cre enzyme. However, due to the recent local epidemic in Fujian, we do not have more time to completely solve the leakage problem of a small part of the Cre enzyme.

Overall circuit: Each part of the experiment has been initially verified, and then the pET30a-pT7-loxP-TT-loxP-gadB-RBS-φX174E plasmid was constructed, and the pET30a-pT7-loxP-TT-loxP-sfGFP plasmid was subjected to PCR to obtain except sfGEP GGA link the gadB-BsaI and RBS-φX174E-BsaI obtained at the beginning to the skeleton to construct the plasmid of pET30a-pT7-loxP-TT-loxP-gadB-RBS-φX174E. After transformation, overnight culture, and cPCR, we saw a bright band at 2443bp, indicating that the plasmid was constructed successfully. Then it was sent to the company for sequencing, the sequencing was correct, and the next experiment was continued.

10.11-10.17

All plasmids have been constructed. This week, the three plasmids pACYCDuet-1-LuxCDABE, Opto-Cre-Vvd, pET30a-pT7-loxP-TT-loxP-gadB-RBS-φX174E will be transferred to BL21(DE3) for co-expression.