IGEM:Tsinghua/2007/Projects/Celcuit

Celcuit

Introduction

Standards in engineering cell-cell communications.

In generally, when engineering prokaryotic cell networks, one of the most difficulties is to isolate signals. On one hand, in the widely-used quorum-sensing system, the quorum-sensing signal is excreted out of the cells, which in spite the concentration gradient in space broadcasts an identical signal to every recipient cell. Therefore, cells with same receptors will be triggered simultaneously. And when trying to engineering a more complex system, the efforts are generally limited by available kinds of signal producers and receptors. On the other hand, 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 various combinations of signals. While the excreted broadcasting prokaryotic signals are unable to work in this way. 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.

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.

It is reported that during conjugation, relaxases can transport fusion proteins to recipient cells, 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 allowing transport of multiple copies of the signals to amplify the signals' effects. 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 that can be easily fused to the transport can be easily engineered. Third, the protein signal is produced in donor cells and degraded by the receptor cells, which allow reutilization of the same signals. This means that the cellular network can be reset to the initial condition. At last, it is facile to find various kinds of protein signals.

Prokaryotic tow hybrid system is a large pool of specific signals.

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

Strain Source E. coli TOP10 Transgen biotech company, Beijing. E. coli S17-1 Prof. Chen's lab collection E. coli JM109 Prof. Chen's lab collection

Plasmids

Plasmid Source pEASY-Blunt Transgen biotech company, Beijing. pBBR1MCS Prof. Chen's lab collection pBluescript II SK(-) Prof. Chen's lab collection pMD18-T Takara, Japan. pLZZGPp Prof. Chen's lab collection

Flowchart

The initial part of the project pursuits the verification of relaxase and C1

transport.

Eperiment 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	Zhenyu Shi, Yuan Zhao, Jing Ma
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	Zhenyu Shi, Jing Ma, Yuan Zhao,
3	7-9	2	3-1	PCR clone the lambda and rnnB terminators in large system.	Success.	Extract the products.	7-9	Zhenyu Shi, Yuan Zhao, Jing Ma
4	7-9	3	3-1	Extract the PCR product.	Success.	Perform Overlap extention PCR.	7-10	Zhenyu Shi, Yuan Zhao, Jing Ma
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	Zhenyu Shi, Yuan Zhao, Jing Ma
6	7-10	5	3-3	Overlap extension PCR to clone in large system.	Success.	Extract the products.	7-10	Zhenyu Shi, Yuan Zhao, Jing Ma
7	7-10	6	3-3	Extract the PCR product.	Success.	Insert the product into pEASY-B cloning vector.	wait for FLP.	Zhenyu Shi, Yuan Zhao, Jing Ma
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	Zhenyu Shi, Yuan Zhao, Jing Ma
9	7-13	8	3-4	PCR screen the clones.	Success.	Culture the right clones for sequencing.	7-14	Zhenyu Shi, Yuan Zhao, Jing Ma
10	7-14	9	3-4	culture the right clones for enzyme digestion verification.	-	Send the cultures for sequencing.	7-15	Zhenyu Shi, Yuan Zhao, Jing Ma
11	7-15	10	3-4	Store the cultures of different clones.		Extract the plasmids for verification and send for sequencing.	7-16	Zhenyu Shi, Yuan Zhao, Jing Ma
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	Zhenyu Shi, Yuan Zhao, Jing Ma
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	Zhenyu Shi, Yuan Zhao, Jing Ma
14	7-16	13	3-4	Send the cultures for sequencing.	-	Wait for sequencing results.	-	Zhenyu Shi, Yuan Zhao, Jing Ma
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	Zhenyu Shi, Yuan Zhao, Jing Ma
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	Zhenyu Shi, Yuan Zhao, Jing Ma
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	Zhenyu Shi, Yuan Zhao, Jing Ma
18	7-22	17	3-4	Order another box of the pEASY-B vector.	-			Zhenyu Shi, Yuan Zhao, Jing Ma
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.		Zhenyu Shi, Yuan Zhao, Jing Ma
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	Zhenyu Shi, Yuan Zhao, Jing Ma
21	7-26	20	3-4	Insert the PCR product into pEASY-B cloning vector. Transformation.	-	Pick clones for PCR screening.	7-27	Zhenyu Shi, Yuan Zhao, Jing Ma
22	7-27	21	3-4	PCR screen the clones.	2 clones were found to be correct.	Culture the clones for sequencing.	7-28	Zhenyu Shi, Yuan Zhao, Jing Ma
23	7-28	22	3-4	Culture the correct clones.	-	Send for sequencing.	7-29	Zhenyu Shi, Han Li, Yuting Wang
24	7-29	23	3-4	Send the culture for sequencing.	-	wait for the sequencing results.	-	Zhenyu Shi, Han Li, Yuting Wang
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	Zhenyu Shi, Han Li, Yuting Wang

Thread 3: 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	Zhenyu Shi, Yuan Zhao, Jing Ma
2	7-12	-	4-1	PCR clone the FLP gene directly from the culture.	Success.	Extract the PCR product.	7-12	Zhenyu Shi, Yuan Zhao, Jing Ma
3	7-12	2	4-1	Extract the PCR product.	Success.	Insert the product into pEASY-B cloning vector.	7-12	Zhenyu Shi, Yuan Zhao, Jing Ma
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	Zhenyu Shi, Yuan Zhao, Jing Ma
5	7-13	4	4-1	PCR screen the clones.	Success.	Culture the right clones for sequencing.	7-14	Zhenyu Shi, Yuan Zhao, Jing Ma
6	7-14	5	4-1	culture the right clones for sequencing.	-	Send the cultures for sequencing.	7-15	Zhenyu Shi, Yuan Zhao, Jing Ma
7	7-15	6	4-1	Store the cultures of different clones.		Extract the plasmids for verification and send for sequencing.	7-16	Zhenyu Shi, Yuan Zhao, Jing Ma
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	Zhenyu Shi, Yuan Zhao, Jing Ma
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	Zhenyu Shi, Yuan Zhao, Jing Ma
10	7-16	9	4-1	Send the cultures for sequencing.	-	Wait for sequencing results.	-	Zhenyu Shi, Yuan Zhao, Jing Ma
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	Zhenyu Shi, Yuan Zhao, Jing Ma
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	Zhenyu Shi, Yuan Zhao, Jing Ma
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	Zhenyu Shi, Yuan Zhao, Jing Ma
14	7-22	13	4-1	Order another box of the pEASY-B vector.	-			Zhenyu Shi, Yuan Zhao, Jing Ma
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	Zhenyu Shi, Yuan Zhao, Jing Ma
16	7-26	15	4-1	PCR clone the LFP gene.	Success.	Insert the PCR product into pEASY-B vector.	7-26	Zhenyu Shi, Yuan Zhao, Jing Ma
17	7-26	16	4-1	Insert the PCR product into pEASY-B cloning vector. Transformation.	-	Pick clones for PCR screening.	7-27	Zhenyu Shi, Yuan Zhao, Jing Ma
18	7-27	17	4-1	PCR screen the clones.	2 clones were found to be correct.	Culture the clones for sequencing.	7-28	Zhenyu Shi, Yuan Zhao, Jing Ma
19	7-28	18	4-1	Culture the correct clones.	-	Send for sequencing.	7-29	Zhenyu Shi, Han Li, Yuting Wang
20	7-29	19	4-1	Send the culture for sequencing.	-	wait for the sequencing results.	-	Zhenyu Shi, Han Li, Yuting Wang
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	Zhenyu Shi, Han Li, Yuting Wang