Part:BBa_K3734001
CHREBP promoter
1.Pattern diagram
Fig.1 The model diagram of CHREBP-LUC
2.Experiment
2.1 Method
We used LUC as report genes to reflect the level of expression through detecting luminescence value at 560nm wavelength and the error REN luminescence caused by the number of eliminated cells.
2.2 Result
CHERBP promoter is induced by glucose and the expression rises along with the raise of glucose concentration.
Fig.2 Electrophoretic diagram of CHREBP PCR product
Fig.3 The expression level of inducible promoter CHREBP was respectively analyzed at 48h in 25mM, 5.6mm and 0mM glucose culture
To eliminate the effect of residual glucose in the medium during transmission, we have conducted experiments in a 72-hour group, the result fits our anticipations more.
Fig.4 The expression level of inducible promoter CHREBP was respectively analyzed at 72h in 25mM, 5.6mm and 0mM glucose culture
3.Caution
Due to the relatively long sequence of CHREBP promoter, DH5α receptor cells containing recombinant enzymes are difficult to meet the
requirements during cloning, and receptor cells with recombinant enzymes may
need to be removed.
Meanwhile, we hope that we can shorten the length of the sequence to improve it although it can work quite well.
Reference：
[1]Jian Meng, Ming Feng, Weibing Dong.Identification of HNF-4α as a key transcription factor to promote ChREBP expression in response to glucose[J].Sci Rep. 2016 Mar 31;6:23944.
GIP promoter
GIP promoter is anticipated in current literature that it can only be used in several certain types of cells like intestine cells, but we proved that it can function will in 293T cells which are usually used in labs.
1.Pattern diagram
Fig.1 The model diagram of CHREBP-LUC
2.Experiments
2.1 Method
We use LUC as report gene, test its real-time luminescence value at 560nm wavelength with specific devices and detect the expression level under different glucose concentration.
2.2 Result
GIP promoter is induced by glucose and the expression level rises along with the raise of glucose concentration.
Fig.2 Changes of LUC expression over time after blue light irradiation with different glucose concentrations
3.Caution
Use medium without glucose when paving in case of the effect caused by glucose residue
Reference：
[1]Anthony T. Cheung,Bama Dayanandan,Jamie T. LewisGlucose-Dependent Insulin Release from Genetically Engineered K Cells[J].Science.2000,290:1959-1962
GI
GI is a photosensitive protein, under the exposure of 450nm blue light, its structure will alter and can form a dimer with LOV. We used GI as a part of our blue light switch.
1.Pattern diagram
Fig.1 The model diagram of GIP-miR21T-GI-GAL4-4XmiR21TC
2.Caution
The duration of the combination of GI and LOV after the blue light exposure will alter as the illuminate method and the intensity of the blue light differ. We used strong light for a 30min exposure and then use weak light for another 30min exposure.
Reference：
[1]Masayuki Yazawa , Amir M Sadaghiani, Brian Hsueh.Induction of protein-protein interactions in live cells using light[J].Nat Biotechnol. 2009 Oct;27(10):941-5.
LOV
LOV is a photosensitive protein, the structure will alter under 450nm blue light exposure and form a dimer with GI. We used LOV as a part of our blue light switch.
1.Pattern diagram
Fig.1 The model diagram of LOV-VP16
2.Caution
The duration of the combination of GI and LOV after the blue light exposure will alter as the illuminate method and the intensity of the blue light differ. We used strong light for a 30min exposure and then use weak light for another 30min exposure.
Reference：
[1]Masayuki Yazawa , Amir M Sadaghiani, Brian Hsueh.Induction of protein-protein interactions in live cells using light[J].Nat Biotechnol. 2009 Oct;27(10):941-5.
GAL4
GAL4 is a common part in double yeast hybrid system. It can recognize and combine with UAS. Because GAL4 is originated from yeast cells, the activation domain can not work in mammalian cells. Therefore, we only used its combining domain and use it together with VP16 which is an activation domain comes from virus to realize the activation of downstream UAS transcription
1.Pattern diagram
Fig.1 The model diagram of GIP-miR21T-GI-GAL4-4XmiR21T
2.Caution
GAL4 combining domain is universal among eukaryotic cells, but the activation factor needs to be chosen differently according to the chassis creature.
Reference：
[1]Chun Jeih Ryu , Charles E Whitehurst, Jianzhu Chen.Expression of Gal4-VP16 and Gal4-DNA binding domain under the control of the T lymphocyte-specific lck proximal promoter in transgenic mice[J].BMB Rep. 2008 Aug 31;41(8):575-80.
VP16
VP16 is a transcription activation factor, also commonly used in double yeast hybrid system. It can realize the activation of downstream UAS transcription. It is usually used together with GAL4 combining domain, which can recognize and combine with UAS, to locate UAS.
1.Pattern diagram
Fig.1 The model diagram of LOV-VP16
2.Caution
GAL4 combining domain is universal among eukaryotic cells, but the activation factor needs to be chosen differently according to the chassis creature.
Reference：
[1]Chun Jeih Ryu , Charles E Whitehurst, Jianzhu Chen.Expression of Gal4-VP16 and Gal4-DNA binding domain under the control of the T lymphocyte-specific lck proximal promoter in transgenic mice[J].BMB Rep. 2008 Aug 31;41(8):575-80.
INSR
Insulin receptor (INSR)is a membrane protein that bid to insulin, feel insullin concentration and activate the downstream MAPK phosphorylation pathway. We have designed a carrier CMV-INSR-EF1A-ZsGreen containing INSR to express INSR, and its expression can be characterized by the green fluorescent protein ZsGreen. Experiments have proved that INSR is successfully expressed in our engineering cells.
1.Experiment
1.1 Method
Observation of green fluorescent protein ZsGreen under laser confocal microscope.
1.2 Result
Fig.1 Electrophoretic diagram of INSR PCR product
INSR is successfully expressed and working in cell 293T
Fig.2 Green fluorescence of INSR expression
2.Caution
Because the INSR sequence is very long, it is difficult for DH5α receptor cells containing recombinant enzymes to meet the requirements during cloning, and receptor cells of recombinant enzymes may need to be removed.
TetR
TetR can be identified and combined with TRE, which is an important part of the Tet-off system. In the absence of tetracycline, TetR binds to TRE. When tetracycline exists, the binding of TetR and TRE will be blocked and the Tet-off system will be shut down.
1.Pattern diagram
Fig.1 The model diagram TetR-ELK
2.Experiment
2.1 Method
After TetR binds to TRE, phosphorylation ELK activates downstream reporting gene mCherry expression, and we observe the expression with laser confocal microscope.
2.2 Results
Fig.2 Under Laser confocal microscopy, fluorescence of mCherry expression downstream of Tet-Off system
3.Caution
If the green fluorescent protein is expressed too much, and if the green fluorescence is too strong, it may cause a series of colors. In order to ensure the accuracy of the experimental results, the level of noise reduction should be raised.
Reference：
[1]Haifeng Ye, Mingqi Xie, Shuai Xue.Self-adjusting synthetic gene circuit for correcting insulin resistance[J].Nat Biomed Eng.2017 Jan;1(1):0005.
ELK
ELK is generally dormant, when INSR accepts insulin to activate the MAPK pathway, Elk is phosphated into an active state, which can activate the expression of the gene downstream TRE.
1.Pattern diagram
Fig.1 The model diagram TetR-ELK
2.Experiments
2.1 Method
We used Western Blot to detect phosphorylation of ERK1/ERK2 in the phosphorylation pathway with ERK antibodies.
2.2 Results
Fig.2 ERK phosphorylation changes with different insulin treatment
Fig.3 ERK phosphorylation changes with different insulin treatment
3.Caution
Despite the length and the complication of phosphorylation pathway, the phosphorylation pathway of protein is a very short process, it is usually completed within minutes even seconds. The time of cracking the cells and collecting protein must be controlled precisely when detecting.
Reference：
[1]Haifeng Ye, Mingqi Xie, Shuai Xue.Self-adjusting synthetic gene circuit for correcting insulin resistance[J].Nat Biomed Eng.2017 Jan;1(1):0005.
TRE
TRE is a DNA structure domain that combined with TetR. It is a part of Tet-off system. Our first purpose of using it is to make phosphated ELK1 to locate and activate the expression of downstream genes (miRNA). The second reason we used it is because of the Tet-off system consists of TetR and TRE can be blocked by tetracycline. Tetracycline will stop the combination between TetR and TRE, stop the system from working. This provides a mandatory brake system for our loop. We can make sure the security and prevent the hypoglycemia caused by abnormal feedback through exogenous tetracycline.
1.Pattern diagram
Fig.1 The model diagram of TRE's work
2.Experiment
2.1 Method
Phosphated ELK will activate the expression of downstream report gene mCherry after the combination of TRE and TetR. We used lase confocal microscope to observe the expression.
2.2 Results
TRE works well.
Fig.2 Under Laser confocal microscopy, fluorescence of mCherry expression downstream of Tet-Off system
3.Caution
If the green fluorescent protein is expressed too much, and if the green fluorescence is too strong, it may cause a series of colors. In order to ensure the accuracy of the experimental results, the level of noise reduction should be raised.
Reference：
[1]Haifeng Ye, Mingqi Xie, Shuai Xue.Self-adjusting synthetic gene circuit for correcting insulin resistance[J].Nat Biomed Eng.2017 Jan;1(1):0005.
mCherry
mCherry is a monomer red fluorescent protein. Experiments proved that when it is combined with exogenous protein on both C side and N side, the activity of fluorescent and the function of combined proteinare not effected and it can be used together with multi types of fluorescent proteins. Due to the phosphorylation pathway is relatively long, we have added mCherry at the bottom as report gene to prove that our pathway works and is regulated by insulin.
1.Pattern diagram
Fig.1 The model diagram of TRE-mCherry-miR21
2.Experiment
2.1 Method
Phosphated ELK will activate the expression of downstream report gene mCherry after the combination of TRE and TetR. We used lase confocal microscope to observe the expression.
2.2 Result
mCherry can be expressed and tested successfully
Fig.2 Under Laser confocal microscopy, fluorescence of mCherry expression downstream of Tet-Off system
miR21
We used NO21 miRNA (miR21), it can not only suppress the expression of target gene by targeting miR21T, but also speed the degradation of mRNA up.
1.Pattern diagram
Fig.1 The model diagram of pG-super-miR21
Fig.2 The model diagram of TRE-mCherry-miR21
2.Experiment
2.1 Method
We used LUC as report genes to reflect the level of expression through detecting luminescence value at 560nm wavelength and the error REN luminescence caused by the number of eliminated cells.
2.2 Result
Fig.3 Electrophoretic diagram of miR21 PCR product
At the time of the design experiment, the first 48-hour test reported that the gene LUC/REN ratio was reported, and miR21 inhibited 40% of the expression, but this was not ideal for our design.
Fig.4 miR21 inhibited Luc expression 48h after transfection
Considering that it may be the reason for the continuous accumulation of LUC expression, we did another 24-hour group, and the results proved that miR21 suppressed 90% of the expression, and the experimental results were very in line with our design requirements.
Fig.5 miR21 inhibited Luc expression 48h after transfection
3.Caution
The miR21 we built is a full-length sequence with flank sequences, which requires precursors such as pre and pri to be sheared and folded and matured within the cell. At the beginning of the experiment, we were worried about its inhibition efficiency and built miR21-stemloop, but because its efficiency can reach 90%, which fully meets our requirements, we still use the full length. Interested iGEMers can further test it.
Reference：
[1]Kai Zhang , Xue-Jiao Yang , Ting-Ting Zhang.In situ imaging and interfering Dicer-mediated cleavage process via a versatile molecular beacon probe.Anal ChimActa.2019Nov4;1079:146-152.
LUC
LUC is a reported gene in the luminescence system of fluorease. It comes from fireflies and characterizes the amount of gene expression in the target by detecting the luminescence value of its excited fluorescent hormone at a wavelength of 560nm. Let's take LUC after CHREBP promoter as an example to prove that LUC can complete the task of reporting genes well.
1.Pattern diagram
Fig.1 The model diagram of CHREBP-LUC
2.Experiments
2.1 Method
We used LUC as report genes to reflect the level of expression through detecting luminescence value at 560nm wavelength and the error REN luminescence caused by the number of eliminated cells.
2.2 Results
The experiments proved that LUC can complete the mission of being a report gene well.
Fig.2 The expression level of inducible promoter CHREBP was respectively analyzed at 48h in 25mM, 5.6mm and 0mM glucose culture
3.Caution
Try to convert REN at the same time to adjust the errors caused by other factors such as different cell numbers and cell states caused by different culture conditions.
Insulin
We have proved that Insulin can be processed and secreted outside the cell in 293T cells.
1.Pattern diagram
Fig.1 The model diagram of 9XUAS-Insulin
2.Experiment
2.1 Method
In the experiment, we used an ELIAS kit specially designed to detect human mature insulin to detect cell eliquidation.
2.2 Results
Insulin can be processed and secreted outside the cell in 293T cells.
Fig.2 Standard curve of absorbance and insulin concentration
Fig.3 Insulin concentration in supernatant after blue light irradiation and dark treatment respectively
Fig.4 Insulin concentration in supernatant (excluding insulin in medium) after blue light irradiation and dark treatment respectively
3.Caution
Insulin is not suitable for long time preservation under the best culture temperature of cells (37℃). In the mean time, cells would also consume some of the expressed insulin. So the best tst time for Insulin is approximately 24 hours after transfection, it is relatively shorter than the test interval of LUC.
Reference：
[1]Mingqi Xie, Haifeng Ye, Hui Wang.β-cell-mimetic designer cells provide closed-loop glycemic control[J].Science.2016 Dec 9;354(6317):1296-1301.
9XUAS
UAS (BBa_K758003) is a piece of small DNA sequence that can be recognized and combined by GAL4 DBD. Team iGEM12_KIT-Kyoto repeated UAS 5 times when using it. In our design, considering of that the mutual effect of our blue light protein would reduce the efficiency and the whole pathway is relatively long, while the react time of our system should be as short as possible, we have made some alterations.
Firstly, we have repeated the UAS before promoter for 9 times. The purpose of constructing 9XUAS-Ad-LUC (BBa_K3734032) and 9XUAS-Ad-insulin(BBa_K3734023) is to enhance the recognition and combination of GAL4 DBD, improve the efficiency of downstream target gene activation.
Secondly, we have used Ad promoter as promoter to make it able to function in eukaryotic cells and improve the expression efficiency after the activation of VP16.
1.Pattern diagram
Fig.1 The model diagram of 9XUAS-Insulin
2.Experiment
2.1 Method
We use LUC as report genes to reflect the level of expression through detecting luminescence value at 560nm wavelength and the error REN luminescence caused by the number of eliminated cells.
2.2 Result
We used the reporting gene LUC as the downstream target gene, and set up a positive control group and a negative control group. The results prove that the efficiency of our improved 9XUAS has been greatly improved, which can meet the requirements of short loop response.
Fig.2 Light controlled system testing experiment
Then we changed the downstream target gene into insulin gene and proved that the insulin can be expressed, processed and secreted successfully and the efficiency of 9XUAS is high enough.
Fig.3: Insulin concentration in supernatant (excluding insulin in medium) after blue light irradiation and dark treatment respectively
3.Caution
Because we have repeated UAS nine times, we should pay attention to avoiding duplicate fragments when designing primer. If you can't avoid duplicate clips, please note the times that UAS in PCR products has been repeated .
Reference：
[1]Chun Jeih Ryu , Charles E Whitehurst, Jianzhu Chen.Expression of Gal4-VP16 and Gal4-DNA binding domain under the control of the T lymphocyte-specific lck proximal promoter in transgenic mice[J].BMB Rep. 2008 Aug 31;41(8):575-80.
GIP-miR21T-GI-GAL4-4XmiR21T
GIP-miR21T-GI-GAL4-4XmiR21T consists of glucose-induced promoter GIP, photosensitive protein GI, double yeast hybrid system components GAL4 and miR21T. It is regulated by both glucose concentration and blue light, which improves the safety of the whole system. At the same time, it is also suppressed by miR21 feedback, which makes the whole system form a closed loop and makes the reaction more sensitive.
In order to increase the safety of patients' use, it may be difficult to achieve the expected goal of accurate regulation by relying on the regulation of blood glucose concentration alone, so we designed a pair of photosensitive proteins of GI (BBa_K3734004) and LOV (BBa_K3734006). Under blue light irradiation, the Protein structure changes and interacts with each other to form a GAL4-GI-LOV-VP16 quadruple, while GAL4 (BBa_K3734005) identifies and combines 9XUAS (BBa_K3734016), so that VP16 can activate the gene of downstream expression.
1.Pattern diagram
Fig.1 The model diagram of GIP-miR21T-GI-GAL4-4XmiR21T
2.Experiment
2.1 Method
We used report gene LUC to represent the effect of GIP-miR21T-GI-GAL4-4XmiR21T
2.2 Result
Fig.2 Light controlled system testing experiment
Fig.3 Expression level analysis under blue light irradiation and dark treatment
3.Caution
The duration of GI and LOV after 450nm blue light exposure will vary with the irradiation method, intensity, etc. In the experiment, we adopted the method of irradiating strong light for 30 minutes first, and then low light for 30 minutes. The GAL4 binding domain is universal in eukaryotic cells, but transcription activators (such as VP16) that cooperate with GAL4 need to be properly selected according to different chassis organisms.
Reference：
[1]Chun Jeih Ryu , Charles E Whitehurst, Jianzhu Chen.Expression of Gal4-VP16 and Gal4-DNA binding domain under the control of the T lymphocyte-specific lck proximal promoter in transgenic mice[J].BMB Rep. 2008 Aug 31;41(8):575-80.
[2]Masayuki Yazawa , Amir M Sadaghiani, Brian Hsueh.Induction of protein-protein interactions in live cells using light[J].Nat Biotechnol. 2009 Oct;27(10):941-5.
[3]Kai Zhang , Xue-Jiao Yang , Ting-Ting Zhang.In situ imaging and interfering Dicer-mediated cleavage process via a versatile molecular beacon probe.Anal ChimActa.2019Nov4;1079:146-152.
LOV-VP16
GLOV is a photosensitive protein, the structure will alter under 450nm blue light exposure and form a dimer with GI. We used LOV as a part of our blue light switch. VP16 is a transcription activation factor, also commonly used in double yeast hybrid system. It can realize the activation of downstream UAS transcription. It is usually used together with GAL4 combining domain, which can recognize and combine with UAS, to locate UAS.
1.Pattern diagram
Fig.1 The model diagram of LOV-VP16
2.Experiment
2.1 Method
We used report gene LUC to represent the effect of LOV-IP16
2.2 Result
Fig.2 Light controlled system testing experiment
Fig.3 Expression level analysis under blue light irradiation and dark treatment
3.Caution
The duration of GI and LOV after 450nm blue light exposure will vary with the irradiation method, intensity, etc. In the experiment, we adopted the method of irradiating strong light for 30 minutes first, and then low light for 30 minutes. The GAL4 binding domain is universal in eukaryotic cells, but transcription activators (such as VP16) that cooperate with GAL4 need to be properly selected according to different chassis organisms.
Reference：
[1]Chun Jeih Ryu , Charles E Whitehurst, Jianzhu Chen.Expression of Gal4-VP16 and Gal4-DNA binding domain under the control of the T lymphocyte-specific lck proximal promoter in transgenic mice[J].BMB Rep. 2008 Aug 31;41(8):575-80.
[2]Masayuki Yazawa , Amir M Sadaghiani, Brian Hsueh.Induction of protein-protein interactions in live cells using light[J].Nat Biotechnol. 2009 Oct;27(10):941-5.
9XUAS-Ad-Insulin
UAS (BBa_K758003) is a piece of small DNA sequence that can be recognized and combined by GAL4 DBD. Team iGEM12_KIT-Kyoto repeated UAS 5 times when using it. In our design, considering of that the mutual effect of our blue light protein would reduce the efficiency and the whole pathway is relatively long, while the react time of our system should be as short as possible, we have made some alterations
Firstly, we have repeated the UAS before promoter for 9 times. The purpose of constructing 9XUAS-Ad-LUC (BBa_K3734032) and 9XUAS-Ad-insulin(BBa_K3734023) is to enhance the recognition and combination of GAL4 DBD, improve the efficiency of downstream target gene activation.
Secondly, we have used Ad promoter as promoter to make it able to function in eukaryotic cells and improve the expression efficiency after the activation of VP16
1.Pattern diagram
Fig.1 The model diagram of 9XUAS-Insulin
2.Experiment
2.1 Method
We used human mature insulin ELISA kits to detect the level of insulin secreted by engineering cells.
2.2 Result
We use the gene of the downstream target with the insulin gene, and successfully proved that insulin can be successfully expressed, processed and secreted out of cells, and 9XUAS is efficient enough.
Fig.2 Insulin and absorbance standard curve
Fig.3 Insulin concentration in supernatant (excluding insulin in medium) after blue light irradiation and dark treatment respectively
3.Caution
Because we repeat UAS nine times, we should pay attention to avoiding duplicate fragments when designing primer. If you can't avoid duplicate clips, please note the times that UAS in PCR products has been repeated.Insulin is not suitable for long time preservation under the best culture temperature of cells (37℃). In the mean time, cells would also consume some of the expressed insulin. So the best tst time for Insulin is approximately 24 hours after transfection, it is relatively shorter than the test interval of LUC.
Reference：
[1]Chun Jeih Ryu , Charles E Whitehurst, Jianzhu Chen.Expression of Gal4-VP16 and Gal4-DNA binding domain under the control of the T lymphocyte-specific lck proximal promoter in transgenic mice[J].BMB Rep. 2008 Aug 31;41(8):575-80.
[2]Mingqi Xie, Haifeng Ye, Hui Wang.β-cell-mimetic designer cells provide closed-loop glycemic control[J].Science.2016 Dec 9;354(6317):1296-1301.
9XUAS-Ad-LUC
UAS (BBa_K758003) is a piece of small DNA sequence that can be recognized and combined by GAL4 DBD. Team iGEM12_KIT-Kyoto repeated UAS 5 times when using it. In our design, considering of that the mutual effect of our blue light protein would reduce the efficiency and the whole pathway is relatively long, while the react time of our system should be as short as possible, we have made some alterations
Firstly, we have repeated the UAS before promoter for 9 times. The purpose of constructing 9XUAS-Ad-LUC (BBa_K3734032) and 9XUAS-Ad-insulin(BBa_K3734023) is to enhance the recognition and combination of GAL4 DBD, improve the efficiency of downstream target gene activation.Firstly, we have repeated the UAS before promoter for 9 times. The purpose of constructing 9XUAS-Ad-LUC (BBa_K3734032) and 9XUAS-Ad-insulin(BBa_K3734023) is to enhance the recognition and combination of GAL4 DBD, improve the efficiency of downstream target gene activation.
Secondly, we have used Ad promoter as promoter to make it able to function in eukaryotic cells and improve the expression efficiency after the activation of VP16
1.Experiment
1.1 Method
We used report gene LUC to represent the working status of 9XUAS-Ad
1.2 Result
we used the reporting gene LUC as the downstream target gene, and set up a positive control group and a negative control group. The results prove that the efficiency of our improved 9XUAS has been greatly improved, which can meet the requirements of short loop response
Fig.1 Light controlled system testing experiment
2.Caution
Because we repeat UAS nine times, we should pay attention to avoiding duplicate fragments when designing primer. If you can't avoid duplicate clips, please note the times that UAS in PCR products has been repeated.
Reference：
[1]Chun Jeih Ryu , Charles E Whitehurst, Jianzhu Chen.Expression of Gal4-VP16 and Gal4-DNA binding domain under the control of the T lymphocyte-specific lck proximal promoter in transgenic mice[J].BMB Rep. 2008 Aug 31;41(8):575-80.
TetR-ELK
TetR can recognize and combine with TRE, it is a important part of Tet-off system. Without tetracycline, TetR can combine with TRE. With the presence of tetracycline, the combination of TetR and TRE will be blocked and the Tet-off system will be shut down. ELK is generally dormant, when INSR receive insulin and activate MAPK, ELK will be phophated and activated, then ELK is able activate expression of target gene downstream TRE.
1.Pattern diagram
Fig.1 The model diagram TetR-ELK
2.Experiment
2.1 Method
After TetR binds to TRE, phosphorylation ELK activates downstream reporting gene mCherry expression, and we observe the expression with laser confocal microscope.
>We also use ERK antibodies to detect phosphorylation of ERK1/ERK2 in the phosphorylation pathway through Western Blot.
2.2 Result
Fig.2 Under Laser confocal microscopy, fluorescence of mCherry expression downstream of Tet-Off system
Fig.3 ERK phosphorylation changes with different insulin treatment
Fig.4 ERK phosphorylation changes with different insulin treatment
3.Caution
Despite the length and the complication of phosphorylation pathway, the phosphorylation pathway of protein is a very short process, it is usually completed within minutes even seconds. The time of cracking the cells and collecting protein must be controlled precisely when detecting
Reference：
[1]Haifeng Ye, Mingqi Xie, Shuai Xue.Self-adjusting synthetic gene circuit for correcting insulin resistance[J].Nat Biomed Eng.2017 Jan;1(1):0005.
TRE-mCherry-miR21
TRE is a DNA structure domain that combined with TetR. It is a part of Tet-off system. This system consists of TetR and TRE can be blocked by tetracycline. Tetracycline will block the combination right between TetR and TRE and stop the system from working
mCherry is a monomer red fluorescent protein. Experiments proved that when it is combined with exogenous protein on both C side and N side, the activity of fluorescent and the function of combined proteinare not effected and it can be used together with multi types of fluorescent proteins. Due to the phosphorylation pathway is relatively long, we have added mCherry at the bottom as report gene to prove that our pathway works and is regulated by insulin.
NO.21 miRNA (miR21) can not only suppress the expression of target gene by targeting miR21T, but also speed the degradation of mRNA up
1.Pattern diagram
Fig.1 The model diagram of TRE-mCherry-miR21
Fig.2 The model diagram of TRE-mCherry-miR21’s work
2.Experiment
2.1 Method
Phosphated ELK will activate the expression of downstream report gene mCherry after the combination of TRE and TetR. We used lase confocal microscope to observe the expression
>As for miR21, we used LUC as report genes to reflect the level of expression through detecting luminescence value at 560nm wavelength and the error REN luminescence caused by the number of eliminated cells
2.2 Result
TRE-mCherry-miR21 works well.
Fig.3 Under Laser confocal microscopy, fluorescence of mCherry expression downstream of Tet-Off system
At the time of the design experiment, the first 48-hour test reported that the gene LUC/REN ratio was reported, and miR21 inhibited 40% of the expression, but this was not ideal for our design.
Fig.4 miR21 inhibited Luc expression 48h after transfection
Considering that it may be the reason for the continuous accumulation of LUC expression, we did another 24-hour group, and the results proved that miR21 suppressed 90% of the expression, and the experimental results were very in line with our design requirements.
Fig.5 miR21 inhibited Luc expression 24h after transfection
3.Caution
If the green fluorescent protein is expressed too much, and if the green fluorescence is too strong, it may cause a series of colors. In order to ensure the accuracy of the experimental results, the level of noise reduction should be raised.
The miR21 we built is a full-length sequence with flank sequences, which requires precursors such as pre and pri to be sheared and folded and matured within the cell. At the beginning of the experiment, we were worried about its inhibition efficiency and built miR21-stemloop, but because its efficiency can reach 90%, which fully meets our requirements, we still use the full length. Interested iGEMers can further test it.
Reference：
[1]Haifeng Ye, Mingqi Xie, Shuai Xue.Self-adjusting synthetic gene circuit for correcting insulin resistance[J].Nat Biomed Eng.2017 Jan;1(1):0005.
[2]Kai Zhang , Xue-Jiao Yang , Ting-Ting Zhang.In situ imaging and interfering Dicer-mediated cleavage process via a versatile molecular beacon probe.Anal ChimActa.2019Nov4;1079:146-152.
CHREBP-LUC
In our experiment, the change of glucose concentration is an important switch of the work loop . CHREBP is only studied as a part of lipid metabolism in current literature. We used it as a glucose-induced promoter and achieved ideal result in cell 293T
LUC is a reported gene in the luminescence system of fluorease. It comes from fireflies and characterizes the amount of gene expression in the target by detecting the luminescence value of its excited fluorescent hormone at a wavelength of 560nm.
1.Pattern diagram
Fig.1 The model diagram of CHREBP-LUC
2.Experiment
2.1 Method
We used LUC as report genes to reflect the level of expression through detecting luminescence value at 560nm wavelength and the error REN luminescence caused by the number of eliminated cells
>2.2 Result
CHERBP promoter is induced by glucose and the expression rises along with the raise of glucose concentration.
TRE-mCherry-miR21 works well.
Fig.2 The expression level of inducible promoter CHREBP was respectively analyzed at 48h in 25mM, 5.6mm and 0mM glucose culture
To eliminate the effect of residual glucose in the medium during transmission, we have conducted experiments in a 72-hour group, the result fits our anticipations more.
Fig.3 The expression level of inducible promoter CHREBP was respectively analyzed at 72h in 25mM, 5.6mm and 0mM glucose culture
3.Caution
Because the INSR sequence is very long, it is difficult for DH5α receptor cells containing recombinant enzymes to meet the requirements during cloning, and receptor cells of recombinant enzymes may need to be removed.
Meanwhile, we hope we can shorten the sequence to improve it although it is good enough. Try to convert REN at the same time to adjust the errors caused by other factors such as different cell numbers and cell states caused by different culture conditions when using LUC.
Reference：
[1]Jian Meng, Ming Feng, Weibing Dong.Identification of HNF-4α as a key transcription factor to promote ChREBP expression in response to glucose[J].Sci Rep. 2016 Mar 31;6:23944.
miR21T-LUC-4XmiR21T
To prove miR21 can target miE21T and suppress the expression of the target gene, we have designed miR21T-LUC-4XmiR21T
1.Pattern diagram
Fig.1 The model diagram of miR21T-LUC-4XmiR21T
2.Experiment
2.1 Method
We used LUC as report genes to reflect the level of expression through detecting luminescence value at 560nm wavelength and the error REN luminescence caused by the number of eliminated cells. In the mean time, we tested the level of mRNA expression through qPCR.
2.2 Result
Fig.2 Electrophoretic diagram of miR21T-LUC-4XmiR21T PCR product
At the time of the design experiment, the first 48-hour test reported that the gene LUC/REN ratio was reported, and miR21 inhibited 40% of the expression, but this was not ideal for our design.
Fig.3 miR21 inhibited Luc expression 48h after transfection
Considering that it may be the reason for the continuous accumulation of LUC expression, we did another 24-hour group, and the results proved that miR21 suppressed 90% of the expression, and the experimental results were very in line with our design requirements.
In the mean time, we have tested miR21 to speed up the degradation of mRNA through qPCR.
Fig.4 miR21 inhibited Luc expression 24h after transfection
Fig.5 miR21 promoted the mRNA degradation efficiency of LUC
3.Caution
Because we have repeated mE21T for 4 times at 3’UTR area, we should avoid the repeated pieces when designing the primer. Please pay attention to how many times does miR21T have repeated in PCR outcome. Meanwhile, there is similar parts between miR21T and padrome structure, which should be paid attention to during the design
Reference：
[1]Kai Zhang , Xue-Jiao Yang , Ting-Ting Zhang.In situ imaging and interfering Dicer-mediated cleavage process via a versatile molecular beacon probe.Anal ChimActa.2019Nov4;1079:146-152.
| None |