IGEM:Tsinghua/2007/Projects/Celcuit
Introduction
Standards in engineering cell-cell communications. |
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When engineering prokaryotic cell networks, one of the most difficulties is isolating signals. For one, it's necessary to get various kinds of signals to allow specific communication. In the widely-used quorum-sensing system, the quorum-sensing signal is excreted out of the cells, which in space broadcasts an identical signal to every recipient cell. In spite of the concentration gradient, that means cells with same receptor will be triggered simultaneously. And when trying to engineering a more complex system, the freedom of design is severely limited by available kinds of quorum-sensing signal producers and receptors. But if a framework allows one signal being isolated from another, it will break the those limits and allow different cells being triggered specifically. For another, space isolation is equally important in engineering. Take the isolated wire cable as an example, although all the wires utilized identical electric current as the medium for signal transport, the isolation allows identical electric current in different wires to generate different combinations of signals. While the excreted broadcasting prokaryotic signals are unable to work in this way because they share the same medium or space. Therefore, those requirements bring the problem posed in the field of bioengineering into a general topic in engineering cell-cell communications. It's necessary to establish a system with standards in cell-cell communication with expected isolation and variance in the kinds of signals. |
Cell-cell communication platform can serve as the framework of cellular circuits. |
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Just take the simple case. If a system allows one cell to deliver specific signal to a specific group of cells, any complicated cellular signal systems can be built up with combinations of a proper number of such systems. It will be also feasible to construct a logical network with such framework. |
Conjugation can perform as cell-cell specific signal pathway. |
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It is reported that relaxases can transport fusion proteins to recipient cells during conjugation, such as Cre. Since relaxases are DNA binding proteins, we further postulated that other DNA binding proteins might also be transported into the recipient cells, which would be a potential useful phenomenon in engineering cell-cell communication. Therefore, both the relaxases and the lambda C1 proteins are chosen as the transport for cell-cell signals during conjugation. Especially, several C1 binding sites can be integrated in one conjugation plasmid to allow the transport of multiple copies of the signals to the recipient cells in order to amplify the effecting. The conjugation system has many advantages. First, the signals are transferred from one cell to another in a point-to-point manner, rather than the excreted broadcast way. Second, protein signals can be easily fused to the DNA-binding transport with pre-constructed system, allowing parallel engineering of many different pairs of signal producer and receptor. Third, the protein signal is produced in donor cells and degraded by the receptor cells. This allows reutilization of the same signals and indicates that the cellular network can be reset to the initial condition for reuse. At last, it is facile to find various kinds of protein signal producer/receptor pairs from two hybrid systems. |
Prokaryotic two hybrid system is a large pool of specific signals. |
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During the last two decades, at least three prokaryotic two hybrid system has been established. If the two hybrid system is integrated with the cell-cell signal transport system, they can provide a large pool for specific signals in cell-cell communications. |
Strains & Plasmids
Strains | ||||||||
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Plasmids | ||||||||||||
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Flowchart
The initial part of the project pursuits the verification of relaxase and C1
transport.
Experiment Records
Thread 1: Celcuit carrier plasmids.
| Nubmer | Date | Inherit | Chart Index | Notes | Results | Plan | Expected Time | Operator |
| 1 | 6-20 | - | 1-1 |
Anneal the phosphated C1 primers and ligate the annealed primers with T4 DNA ligase for 16 hours. |
- | Check the ligation result. | 6-21 | Peng Dong |
| 2 | 6-20 | - | 1-3 |
Extract the pLZZGPp plasmid. Use HindIII to cut the plasmid and add CIAP to dephosphate the digested plasmid. |
- | Recover the digested & dephosphated plasmid. | 6-21 | Peng Dong |
| 3 | 6-21 | 2 | 1-3 | Use DNA extraction kit to purify the digested pLZZGPp plasmid. | Purified digested plasmid. | Ligation. | 6-21 | |
| 4 | 6-21 | 1 | 1-2 | Check the ligation products. |
6 bands were seen in the gel. The annealed C1 primers are ligated into multiple copies. |
Ligation. | 6-21 | Peng Dong |
| 5 | 6-21 | 3,4 | 1-3 |
Add the the purified digested pLZZGPp plasmid to the C1 ligation system to insert the C1 fragments into the pLZZGPp plasmid. The negative control ligation used only the digested pLZZGPp plasmids. |
- | Transformation. | 6-22 | Peng Dong |
| 6 | 6-22 | 5 | 1-3 |
Transformation. The ligation system is transformed into E. coli TOP10 competent cells. |
- | Pick clones for verification. | 6-23 | Peng Dong |
| 7 | 6-23 | 6 | 1-3 | 8 clones on the ligation plate were picked. |
There are 50% more clones on the plate of ligation than on the negative control. |
Store the cultures and send them for sequencing. | 6-24 | Peng Dong |
| 8 | 6-24 | 7 | 1-3 | The 8 clones are stored sent to sequencing. | - | Expect the sequencing results. | 6-30 | Peng Dong |
| 9 | 8-2 | 8 | 1-3 |
The 8 sequencing results are analyzed and unfortunately none of them was found to be desired clones. |
Failed in construction the clones. | Need to restart. | 8-5 | Zhenyu Shi |
Thread 2: Celcuit Initiator.
| Nubmer | Date | Inherit | Chart Index | Notes | Results | Plan | Expected Time | Operator |
| 1 | 4-28 | - | 2-3 |
USE PCR to clone the FLP gene and insert the PCR product into pEASY-B cloning vector. Transform the ligation system. |
- | Pick clones. | 4-29 | Xinyu Zhao |
| 2 | 4-29 | 1 | 2-3 | 4 clones were picked and cultured. | - | Extract plasmid and perform double digestion verification. | 4-30 | Xinyu Zhao |
| 3 | 4-30 | 2 | 2-3 | Extract the plasmid and use double digest to screen. | Two clones are found to be right. | Wait the sequencing result. | 5-4 | Xinyu Zhao |
| 4 | 5-5 | 3 | 2-3 | The sequencing results were analyzed. | Both the two were found to be correct clones. | Wait for other Fragments and the expression plasmid. | - | Zhenyu Shi |
| 5 | 5-5 | - | 2-1 |
USE PCR to clone the lambda C1 gene and insert the PCR product into pEASY-B cloning vector. Transform the ligation system. |
- | Pick clones. | Zhenyu Shi | |
| 6 | 5-5 | - | 2-2 |
USE PCR to clone the Mob gene and insert the PCR product into pEASY-B cloning vector. Transform the ligation system. |
- | Pick clones. | Zhenyu Shi | |
| 7 | 5-6 | 5,6 | 2-1,2-2 | 4 clones of both lambda C1 and Mob are picked and use PCR to screened. | All the clones are found to be posive in PCR screen. | Send the clones for sequencing. | 5-7 | Zhenyu Shi |
| 8 | 5-8 | 7 | 2-1,2-2 | The clones are sent for sequencing. | - | Wait the sequencing results. | 5-12 | Zhenyu Shi |
| 9 | 5-15 | 8 | 2-1,2-2 | The A Mob clone and a C1 clone were found to be correct. | - | Wait for the expression vector. | - | Zhenyu Shi |
Thread 3: Celcuit Reporter.
| Nubmer | Date | Inherit | Chart Index | Notes | Results | Plan | Expected Time | Operator |
| 1 | 7-9 | - | 3-1 | Culture the BW25141 harboring pKD13 plasmids. | - | Extract the plasmids. Store the strain. | 7-10 | Yuan Zhao, Jing Ma, Zhenyu Shi |
| 2 | 7-9 | - | 3-1 |
PCR clone the lambda and rnnB terminators directly from the culture of BW25141. |
The two terminators are successfully cloned. |
Enlarge the PCR system and extract the PCR product for further overlap extention PCR. |
7-9 | Yuan Zhao, Jing Ma, Zhenyu Shi |
| 3 | 7-9 | 2 | 3-1 | PCR clone the lambda and rnnB terminators in large system. | Success. | Extract the products. | 7-9 | Yuan Zhao, Jing Ma, Zhenyu Shi |
| 4 | 7-9 | 3 | 3-1 | Extract the PCR product. | Success. | Perform Overlap extention PCR. | 7-10 | Yuan Zhao, Jing Ma, Zhenyu Shi |
| 5 | 7-10 | 4 | 3-2,3-3 |
Anneal the two PCR products and use gradient overlap extension PCR to optimize the conditions. |
Success. | Use the optimal condition to clone the Products. | 7-10 | Yuan Zhao, Jing Ma, Zhenyu Shi |
| 6 | 7-10 | 5 | 3-3 | Overlap extension PCR to clone in large system. | Success. | Extract the products. | 7-10 | Yuan Zhao, Jing Ma, Zhenyu Shi |
| 7 | 7-10 | 6 | 3-3 | Extract the PCR product. | Success. | Insert the product into pEASY-B cloning vector. | wait for FLP. | Yuan Zhao, Jing Ma, Zhenyu Shi |
| 8 | 7-12 | 7 | 3-4 | Insert PCR product to pEASY-B vector. Transformation | - | Pick clones from the plate amd perform PCR screening. | 7-13 | Yuan Zhao, Jing Ma, Zhenyu Shi |
| 9 | 7-13 | 8 | 3-4 | PCR screen the clones. | Success. | Culture the right clones for sequencing. | 7-14 | Yuan Zhao, Jing Ma, Zhenyu Shi |
| 10 | 7-14 | 9 | 3-4 | culture the right clones for enzyme digestion verification. | - | Send the cultures for sequencing. | 7-15 | Yuan Zhao, Jing Ma, Zhenyu Shi |
| 11 | 7-15 | 10 | 3-4 | Store the cultures of different clones. | Extract the plasmids for verification and send for sequencing. | 7-16 | Yuan Zhao, Jing Ma, Zhenyu Shi | |
| 12 | 7-16 | 11 | 3-4 |
Enzyme digestion of the plamids with (XhoI & SalI) for the positive screening and with (SalI & SpeI) for the negative screening. |
Failed. None of the digest turn out to show right DNA strips. | Send for sequencing. Use different enzyme sites. | 7-16 | Yuan Zhao, Jing Ma, Zhenyu Shi |
| 13 | 7-16 | 12 | 3-4 | Enzyme digestino with Fermentas XhoI & SalI. | Failed. Maybe the cloning vector is wrong. | Send the cultures for sequencing. | 7-16 | Yuan Zhao, Jing Ma, Zhenyu Shi |
| 14 | 7-16 | 13 | 3-4 | Send the cultures for sequencing. | - | Wait for sequencing results. | - | Yuan Zhao, Jing Ma, Zhenyu Shi |
| 15 | 7-19 | 14 | 3-4 |
The sequencing results are analyzed and 2 clones were found to be right clones. But the restriction enzyme sites on the vector were not shown in sequencing results. It's even more possible that the cloning vector is wrong. |
- | Culture the rights clones for verification. | 7-17 | Yuan Zhao, Jing Ma, Zhenyu Shi |
| 16 | 7-20 | 15 | 3-4 | Extract the plasmid and perform enzyme digestion for 8 hours. | None of the clones were correctly digested. | Digest longer lest the digestion is not completed. | 7-21 | Yuan Zhao, Jing Ma, Zhenyu Shi |
| 17 | 7-21 | 16 | 3-4 | Check the digestion products. | None of the plasmids from those clones were digested. | Reorder another box of pEASY-B cloning vector. | 7-22 | Yuan Zhao, Jing Ma, Zhenyu Shi |
| 18 | 7-22 | 17 | 3-4 | Order another box of the pEASY-B vector. | - | Yuan Zhao, Jing Ma, Zhenyu Shi | ||
| 19 | 7-25 | 18 | 3-4 |
The transgene company comfirmed that the previous pEASY-B vector was wrong. |
- | Repeat the cloning of the lambda-rrnB terminators. | Yuan Zhao, Jing Ma, Zhenyu Shi | |
| 20 | 7-26 | 19 | 3-1,3-2,3-3 | PCR clone the lambda-rrnB terminators. | Success. | Insert the PCR product into pEASY-B vector. | 7-26 | Yuan Zhao, Jing Ma, Zhenyu Shi |
| 21 | 7-26 | 20 | 3-4 | Insert the PCR product into pEASY-B cloning vector. Transformation. | - | Pick clones for PCR screening. | 7-27 | Yuan Zhao, Jing Ma, Zhenyu Shi |
| 22 | 7-27 | 21 | 3-4 | PCR screen the clones. | 2 clones were found to be correct. | Culture the clones for sequencing. | 7-28 | Yuan Zhao, Jing Ma, Zhenyu Shi |
| 23 | 7-28 | 22 | 3-4 | Culture the correct clones. | - | Send for sequencing. | 7-29 | Yuan Zhao, Jing Ma, Zhenyu Shi |
| 24 | 7-29 | 23 | 3-4 | Send the culture for sequencing. | - | wait for the sequencing results. | - | Yuan Zhao, Jing Ma, Zhenyu Shi |
| 25 | 8-4 | 24 | 3-4 |
The sequencing results were analyzed and both 2 clones were found to be correct. |
- | Culture the correct clones. | 8-5 | Yuan Zhao, Jing Ma, Zhenyu Shi |
Thread 4: Celcuit FLP verifier.
| Nubmer | Date | Inherit | Chart Index | Notes | Results | Plan | Expected Time | Operator |
| 1 | 7-12 | - | 4-1 | Culture the plasmid harboring FLP gene. | - | Extract the plasmid. | 7-13 | Yuan Zhao, Jing Ma, Zhenyu Shi |
| 2 | 7-12 | - | 4-1 | PCR clone the FLP gene directly from the culture. | Success. | Extract the PCR product. | 7-12 | Yuan Zhao, Jing Ma, Zhenyu Shi |
| 3 | 7-12 | 2 | 4-1 | Extract the PCR product. | Success. | Insert the product into pEASY-B cloning vector. | 7-12 | Yuan Zhao, Jing Ma, Zhenyu Shi |
| 4 | 7-12 | 3 | 4-1 | Insert the FLP PCR product into pEASY-B plasmid. Transformation. | - | Pick clones from the plate amd perform PCR screening. | 7-13 | Yuan Zhao, Jing Ma, Zhenyu Shi |
| 5 | 7-13 | 4 | 4-1 | PCR screen the clones. | Success. | Culture the right clones for sequencing. | 7-14 | Yuan Zhao, Jing Ma, Zhenyu Shi |
| 6 | 7-14 | 5 | 4-1 | culture the right clones for sequencing. | - | Send the cultures for sequencing. | 7-15 | Yuan Zhao, Jing Ma, Zhenyu Shi |
| 7 | 7-15 | 6 | 4-1 | Store the cultures of different clones. | Extract the plasmids for verification and send for sequencing. | 7-16 | Yuan Zhao, Jing Ma, Zhenyu Shi | |
| 8 | 7-16 | 7 | 4-1 |
Enzyme digestion of the plamids with (SacI & XbaI) for the positive screening. |
Failed. None of the digest turn out to show right DNA strips. | Send for sequencing. Use different enzyme sites. | 7-16 | Yuan Zhao, Jing Ma, Zhenyu Shi |
| 9 | 7-16 | 8 | 4-1 | Enzyme digestino with Fermentas SacI & XbaI. | Failed. Maybe the cloning vector is wrong. | Send the cultures for sequencing. | 7-16 | Yuan Zhao, Jing Ma, Zhenyu Shi |
| 10 | 7-16 | 9 | 4-1 | Send the cultures for sequencing. | - | Wait for sequencing results. | - | Yuan Zhao, Jing Ma, Zhenyu Shi |
| 11 | 7-19 | 10 | 4-1 |
The sequencing results are analyzed and 3 clones were found to be right clones. But the restriction enzyme sites on the vector were not shown in sequencing results. It's even more possible that the cloning vector is wrong. |
- | Culture the rights clones for verification. | 7-17 | Yuan Zhao, Jing Ma, Zhenyu Shi |
| 12 | 7-20 | 11 | 4-1 | Extract the plasmid and perform enzyme digestion for 8 hours. | None of the clones were correctly digested. | Digest longer lest the digestion is not completed. | 7-21 | Yuan Zhao, Jing Ma, Zhenyu Shi |
| 13 | 7-21 | 12 | 4-1 | Check the digestion products. | None of the plasmids from those clones were digested. | Reorder another box of pEASY-B cloning vector. | 7-22 | Yuan Zhao, Jing Ma, Zhenyu Shi |
| 14 | 7-22 | 13 | 4-1 | Order another box of the pEASY-B vector. | - | Yuan Zhao, Jing Ma, Zhenyu Shi | ||
| 15 | 7-25 | 14 | 4-1 |
The transgene company comfirmed that the previous pEASY-B vector was wrong. |
- | Repeat the cloning of the FLP gene. | 7-26 | Yuan Zhao, Jing Ma, Zhenyu Shi |
| 16 | 7-26 | 15 | 4-1 | PCR clone the LFP gene. | Success. | Insert the PCR product into pEASY-B vector. | 7-26 | Yuan Zhao, Jing Ma, Zhenyu Shi |
| 17 | 7-26 | 16 | 4-1 | Insert the PCR product into pEASY-B cloning vector. Transformation. | - | Pick clones for PCR screening. | 7-27 | Yuan Zhao, Jing Ma, Zhenyu Shi |
| 18 | 7-27 | 17 | 4-1 | PCR screen the clones. | 2 clones were found to be correct. | Culture the clones for sequencing. | 7-28 | Yuan Zhao, Jing Ma, Zhenyu Shi |
| 19 | 7-28 | 18 | 4-1 | Culture the correct clones. | - | Send for sequencing. | 7-29 | Han Li, Yuting Wang, Zhenyu Shi |
| 20 | 7-29 | 19 | 4-1 | Send the culture for sequencing. | - | wait for the sequencing results. | - | Han Li, Yuting Wang, Zhenyu Shi |
| 21 | 8-3 | 20 | 4-1 |
The sequencing results were analyzed and both 2 clones were found to be correct. |
- | Culture the correct clones. | 8-3 | Han Li, Yuting Wang, Zhenyu Shi |
Project Staff
Dry Experiment |
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Wet Experiment |
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