Difference between revisions of "Part:BBa K2243000"
Line 1: Line 1:
 +
<partinfo>BBa_K2707007 short</partinfo>
 +
<h2> 1．Usage </h2>
 +
<b> 1.1 Briefing on PTRCCS<br></b>
 +
PTRCCS, namely the Plasmid Tightly Regulated Copy-Control System in pGF (plasmid Genome Fast) Vector [1], can help the
 +
artificially synthesized genomes achieve stable replication in E. coli, and tightly control the copy number conversion
 +
of the synthetic genome in the E. coli - EPI300 strain, converting the single copy into the copy number of up to 100.
 +
It has been reported that a variety of artificially synthesized genomes, such as Saccharomyces cerevisiae genome [2]
 +
and plant virus genome [3], have been successfully synthesized using vectors containing this system. <br>
  
−
<partinfo>BBa_K2243000 short</partinfo>
+
<b> 1.2 The significance of PTRCCS to our project <br></b>
−
<h2>Improvement</h2>
+
<b> This year, we successfully employed this system to convert the assembled mitochondrial genomes of S. cerevisiae into E.coli for stable cloning.</b> <br>
 +
In the course of our experiments, there has been a very serious problem, which was stable genomic clones could not be
 +
obtained in E. coli and random mutations and deletions occurred after the assembly of mitochondrial genomes in
 +
Saccharomyces cerevisiae. Later, with reference to the design of pGF Vector, we added this system to our vector and
 +
successfully obtained the artificial genome synthesized by stable cloning in E. coli. <br>
  
−
This part was improved from BBa_K1132010.
+
<b> 1.3 Providing reference for the teams to participate <br></b>
−
https://parts.igem.org/Part:BBa_K1132010
+
Based on our researches, it is safe to say that we are the first team in all iGEM teams to manually design and
 +
synthesize vital genomes. With consideration of the importance of PTRCCS to the successful competition of our project
 +
this year, we strongly recommend that PTRCCS be used by all teams working on related subject. Good news is that there
 +
are already commercial products based on this system and hopefully the complete vector DNA containing the system will
 +
be soon available. If necessary, please feel free to contact us and we are more than glad to provide the complete
 +
vector containing PTRCCS we used this year. <br>
  
−
<h2>Abstract</h2>
+
<h2>2. Biology</h2>
 +
PTRCCS consists of two parts: (1) ParA-ParB-ParC plasmid partition system; (2) oviS/oviV copy-control system. <br>
 +
<!--这里有张图-->
  
−
TP901-1 integrase comes from TP901-1 phage and can bind to specific attB/P sites to catalyze DNA recombination. It helps the TP901-1 phage to integrate  its genome into bacterial genome naturally.
+
<b>2.1 ParA-ParB-ParC plasmid partition system：</b> <br>
 +
The ParA-ParB-ParC from the F plasmid in E. coli consists of three elements that are essential for plasmid partition:
 +
Protein SopA, Protein SopB and Cis-acting region sopC. The system ensures the proper distribution of newly-replicated
 +
plasmids to daughter cells during cell division, when these proteins mutually impact. [4] <br>
  
−
By constructing the attB/P sites in different directions, TP901-1 can catalyze the recombination of DNA between their sites, leading to inversion when attB/P are in opposite directions and excision when attB/P are in the same directions. TP901-1 is widely used to construct combinational logic gate and performs well in changing DNA sequence.  
+
<b>2.2 oriS/oriV Copy-Control system：</b> <br>
 +
The oriS/oriV Copy-Control system is comprised of the oriS(ori2)-repE-incC system that controls a single copy of the
 +
plasmid and the oriV/(TrfA) system that implements a strictly controllable multicopy. [5] The oriS(ori2)-repE-incC
 +
system derives from F plasmid in E. coli, composed of replicon oriS(ori2), protein repE and incompatibility region
 +
incC. In the single copy mode, plasmid replication initiates at oriS (ori2), which consists of (1) four directly
 +
repeated sequences of 19 bp (iterons), (2) an AT-rich region, and (3) binding sites for the host DnaA protein. The RepE
 +
protein (251 residues, 29 kD), when in the monomeric form, mediates the assembly of a replication complex at oriS. The
 +
dimeric form of RepE binds to the inverted repeats of the repE operator exerting autogenous repression. [6] <br>
 +
The oriV/(TrfA) system derives from RK2 Vector. The oriV origin of replication consists of eight 17- bp direct repeats
 +
(iterons) that bind a monomeric form of the initiation protein TrfA [7]. DNA replication oriV is completely inactive in
 +
the commonly used hosts, because they do not produce the TrfA replication protein upon which replication at oriV
 +
depends. To supply the TrfA protein, Jadwiga Wild and his partner constructed special hosts, in which the synthesis of
 +
copy-up TrfA mutant protein is very tightly controlled by the ParaBAD (PBAD) promoter and AraC protein. [8] <br>
  
−
<h2>Biology</h2>
+
<h2>3. Characterization</h2>
−
TP901-1 recombinase is a serine recombinase enzyme derived from phage TP901-1 of Lactococcus lactis subsp. cremoris. The enzyme uses a topoisomerase like mechanism to carry out site specific recombination events. It (1.5 kDa) is known to integrate DNA fragment between two DNA recognition sites (attB/P site). With the help of its specific Recombination Directionality Factor (RDF) see the tag BBa_K2243014, TP901-1 recombinase can also flip DNA between the attachment sites, which makes the process reversible.  
+
<b>3.1 PTRCCS verified to be useful </b> <br>
 +
We have not been able to convert the assembled mitochondrial genome of S. cerevisiae into E. coli for stable cloning
 +
even after numerous attempts before applying this system. Nonetheless, hardly had we equipped the vectors with this
 +
system when we successfully realized the cloning of the assembled mitochondrial genome in Saccharomyces cerevisiae
 +
which was later transferred into E. coli for stable cloning. <br>
 +
<!-- 最简线粒体基因组胶图、如果拿不到就换成接口验证的图-->
  
−
<html>
+
<b>3.2 Characterization Purpose</b>
−
<body>
+
Jadwiga Wild and his partner pointed out in their work that vectors containing this system were capable of conversion
 +
from single copy mode to multiple copy mode only after induction and the copy number was determined by the length of
 +
the sequences inserted in the vectors. [8] <br>
 +
Therefore, we measured the copy number of the vector containing the system in the case of inserting the minimal S.
 +
cerevisiae genome sequence we designed this year. <br>
 +
We adopted the QPCR method to measure the E. coli plasmid copy number, which was ever carried out by Lee C, Kim J, Shin
 +
S G, et al. [9] <br>
  
−
<img src="https://static.igem.org/mediawiki/2017/0/0b/Peking_flipflop_fig1.svg"  height="400" width="500"/>
+
<!--需要数据--> <div style="text-indent:52px;">
 +
    aljifjaewfjiwajigjiajgilajfijwailfjiwaejfil
 +
</div>
  
−
</body>
+
<b>3.3 Protocol of Copy Number Determination qPCR</b> <br>
−
</html>
+
<b>3.3.1 Lysate standard sample qPCR </b> <br>
−
 
+
1.Inoculate a single colony into 5 mL of liquid LB medium with corresponding antibiotic and incubate in the shaker at 37 °C; <br>
−
 
+
2.After 14-16h of growth, transfer 100 μL of suspended cells to 5 mL of fresh liquid LB medium with corresponding antibiotic and incubate at 37 °C until the OD600 reaches 0.7-0.8;<br>
−
Fig 1. Site-specific recombination either integrates, deletes or reverses a DNA sequence
+
3.Spin down a suspended 1 mL of cells of 0.7 OD600 at 8.0g for 15 min<br>
−
 
+
(Growth conditions are specified at the end of the protocol);<br>
−
 
+
4.Remove the medium and resuspend the cell pellet in 1 mL of PBS;<br>
−
<h2>Usage</h2>
+
5.Spin down the suspended of cells at 8.0 g for 15 min;<br>
−
Many researchers have paid attention to recombinases because of their ability of changing genetic circuits. TP901-1 is one of the recombinases with outstanding performance. In existence of recombinase TP901-1, different orientation of attB and attP allows the sequence to be flipped, excised, or inserted between recognition sites, which makes it useful for gene editing. In our project, we selected TP901-1 to flip the sequence flanked by attB and attP site for Bio-Flip-Flop construction.
+
6.Repeat steps 2 and 3;<br>
−
 
+
7.Completely remove PBS from the cell pellet;<br>
−
<h2>Characterization</h2>
+
8.Incubate cells at 95 °C for 10 min;<br>
−
 
+
9.Store cells at -20 °C for 10 min;<br>
−
Since the viability of a bio-flip-flop relies on the performance of two integrases and their corresponding excisionases. To select integrases for the bio-flip-flop, we constructed expression vectors for different recombinases and tested their performance individually.
+
10.Completely resuspend dry cell pellet in 100 μL of water by pipetting. Then vortex for 30s and spin down;<br>
−
 
+
11.Make an initial dilution by transferring 10 μL of resuspended cell to 40 μL of water. Pipet carefully vortex for 30s and spin down;<br>
−
To make sure that T[901 have an optimal performance. We used the standard testing system, consisting of the integrase expression plasmid and the recombination reporter plasmid (BBa_K2243010). By choosing the vector with different replication origins(a p15A origin with a pTac promoter, and a ColE1 origin with a pBAD promoter) and the RBS sequences upon the integrase, we measure the recombination efficiency under different conditions.The expression vector and reporter of a recombinase were used to co-transform E. coli Top10 and samples were prepared for testing. We picked out our optimal RBS with low leakage and high efficiency for both backbone.
+
12.Make a second dilution by transferring 10 μL of to 90 μL of water. Pipet carefully vortex for 30s and spin down;<br>
−
 
+
13.For X reactions, make two different mixes using chromosome gene and plasmid gene primers:<br>
−
 
+
X*6 μL of water<br>
−
<html>
+
X*1 μL Forward primer 20 uM<br>
−
<body>
+
X*1 μL Reverse primer 20 uM<br>
−
 
+
X*10 μL of SYBR Green<br>
−
<img src="https://static.igem.org/mediawiki/2017/f/fc/Peking_flipflop_fig_5.svg"  height="500" width="600"/>
+
14. First, transfer 18 μL of mix with chromosome primers to first X tubes, then transfer 18 μL of plasmid primers mix to other X tubes (X*2 tubes);<br>
−
 
+
15. Add 2 μL of each diluted sample to the tubes;<br>
−
</body>
+
16. Tenderly close the caps;<br>
−
</html>
+
17. Run the reaction.<br>
−
 
+
−
Figure 2. The standard testing system used to characterize the recombinases.
+
−
 
+
−
 
+
−
<html>
+
−
<body>
+
−
 
+
−
<img src="https://static.igem.org/mediawiki/2017/d/dc/Peking_flipflop_fig_6.svg"  height="400" width="500"/>
+
−
 
+
−
</body>
+
−
</html>
+
−
 
+
−
Fig 3. Integrase expression vectors with different replication origins. The one shown on top has a re-laxed ColE1 replication origin and recombinase expression is induced by arabinose via a pBad induc-tion system. The one shown in the bottom picture has a relaxed p15A replication origin and recom-binase expression is induced by IPTG.
+
−
 
+
−
We used a microplate reader to roughly measure the efficiency of the selected integrases. We used flow cytometry to conduct a more accurate characterization.
+
−
 
+
−
<h3>Microplate Readers</h3>
+
−
 
+
−
Microplate readers are instruments used to detect biological, chemical or physical events in samples in microtiter plates. We used a microplate reader to detect optical density and fluorescence intensity follwing the procedure as below:
+
−
+
−
1.Pick and inoculate single colony into 1ml of LB media with antibiotics in a V-bottom 96-well plate. Grow the culture overnight (about 12 hours) at 37°C, 220 rpm in a Thermo VARIOSKAN FLASH shaker.
+
−
 
+
−
2. Then, an aliquot comprising 2 μL of the culture was transferred into 1ml LB media with antibiotics and inducer (1mM IPTG or 10mM arabinose). Culture overnight (about 12 hours) at 37°C and 220 rpm.  
+
−
 
+
−
3. Aliquot 1 μl of the cultures in the deep-well plates, then copy them to the 96-well plate filled with 200μl PBS. Don’t forget to leave at least 12 wells with only PBS in it as your negative conctrol.
+
−
 
+
−
4. Measure the samples (OD and Fluorescence). I
+
−
The former represents the density of bacteria, and the latter implies the efficiency of recombination.
+
−
 
+
−
 
+
−
For expression vector with p15A replication origin, proper RBS for TP901-1 was picked out.
+
−
 
+
−
<html>
+
−
<body>
+
−
 
+
−
<img src="https://static.igem.org/mediawiki/parts/5/54/TP901-1_Flipping_Efficiency_p15A.png"  height="350" width="400"/>
+
−
 
+
−
</body>
+
−
</html>
+
−
 
+
−
Figure 4. TP901-1 recombination efficiency with variety of RBS from iGEM (B0030~B0035).
+
−
 
+
−
<h3>Flow Cytometry</h3>
+
−
For more detailed measurements,flow cytometry were used to evaluate the recombination efficiency. Our procedures are as follows:
+
−
 
+
−
1.Single colonies were picked and used to inoculate into 1ml of LB media with antibiotics in a V-bottom 96-well plate.
+
−
 
+
−
2.The cultures were grown at 37°C and 1000 RPM for 12h. Subsequently, an aliquot comprising 2 μL of the culture was transferred into 1ml of M9 glucose media with antibiotics and inducer (1mM IPTG or 10mM arabinose for RBS tuning, gradient concentration for transfer curve) in a V-bottom 96-well plate.
+
−
 
+
−
3.The cultures were grown at 37°C and 1000 RPM for 15h. An aliquot comprising 2μL of the cul-ture was transferred into 198 μL of phosphate buffered saline (PBS) containing 2 mg/mL kanamycin in a 96-well plate. This mixture was incubated for one hour at room temperature before testing. Two lasers were used to excite GFP and RFP simultaneously. Single-cell fluorescence distribution at both emission wavelengths was recorded.
+
−
 
+
−
The counted cells were gated to eliminate the population which showed no fluorescence. The remaining cells were divided into two subsets by a diagonal: RFP sub-set and GFP subset. The recombination efficiency was estimated from the proportion of the RFP subset in the total fluorescent population.
+
−
 
+
−
<html>
+
−
<body>
+
−
 
+
−
<img src="https://static.igem.org/mediawiki/2017/b/ba/Peking_flipflop_fig_7.png"  height="250" width="500"/>
+
−
 
+
−
</body>
+
−
</html>
+
−
 
+
−
Fig 5.A example of Gating the RFP and GFP subsets. Change of fluorescence after induction was seen. Left: no inducer. Right: 10 mM Ara for 15h.
+
−
 
+
−
For the vector with ColE1 replication origin, we found proper RBS in a list of calculated RBSs for TP901-1.
+
−
 
+
−
<html>
+
−
<body>
+
−
 
+
−
<img src="https://static.igem.org/mediawiki/2017/0/0b/Peking_flipflop_fig13b.png"  height="350" width="400"/>
+
−
 
+
−
</body>
+
−
</html>
+
−
 
+
−
Figure 6. TP901-1 recombination efficiency with various RBS. T.I.R = Translation Initiation Rate
+
−
 
+
−
<h3>Fusion Protein</h3>
+
−
Besides, we construct integrase-RDF fusion protein to recognize and bind attL and attR sequences,so the state transitions become reversible.click here to read more.
+
−
https://parts.igem.org/Part:BBa_K2243014
+
−
 
+
−
<h3>Inversion Efficiency</h3>
+
−
Furthermore, we co-transformed the system containing the terminator flanked by TP901-1 attB/P sites and the system expressing the TP901-1 recombinase to test the inversion efficiency using the plate reader. And the results are show in the third bar group in the graph below.
+
−
 
+
−
[[File:Peking 3rdG inversion.png|400px|thumb|center|Fig.7 Terminators flanked by two pair of attB/P sites Induced with 0.1M IPTG and 10mM Arabinose]]
+
−
 
+
−
<h2>Reference</h2>
+
−
1.Baker, T. A., Bell, S. P., Gann, A., Levine, M., & Losick, R. (1970). Molecular biology of the gene.
+
−
 
+
−
2.Roquet, Nathaniel et al. "Synthetic recombinase-based state machines in living cells." Science 353.6297 (2016): aad8559.
+
−
 
+
−
3.Bonnet, J., Yin, P., Ortiz, M. E., Subsoontorn, P., & Endy, D. (2013). Amplifying genetic logic gates. Science, 340(6132), 599-603.
+

Revision as of 08:53, 16 October 2018

Plasmid Tightly Regulated Copy-Control System

1．Usage

1.1 Briefing on PTRCCS
 PTRCCS, namely the Plasmid Tightly Regulated Copy-Control System in pGF (plasmid Genome Fast) Vector [1], can help the artificially synthesized genomes achieve stable replication in E. coli, and tightly control the copy number conversion of the synthetic genome in the E. coli - EPI300 strain, converting the single copy into the copy number of up to 100. It has been reported that a variety of artificially synthesized genomes, such as Saccharomyces cerevisiae genome [2] and plant virus genome [3], have been successfully synthesized using vectors containing this system.

1.2 The significance of PTRCCS to our project
This year, we successfully employed this system to convert the assembled mitochondrial genomes of S. cerevisiae into E.coli for stable cloning.
In the course of our experiments, there has been a very serious problem, which was stable genomic clones could not be obtained in E. coli and random mutations and deletions occurred after the assembly of mitochondrial genomes in Saccharomyces cerevisiae. Later, with reference to the design of pGF Vector, we added this system to our vector and successfully obtained the artificial genome synthesized by stable cloning in E. coli.

1.3 Providing reference for the teams to participate
Based on our researches, it is safe to say that we are the first team in all iGEM teams to manually design and synthesize vital genomes. With consideration of the importance of PTRCCS to the successful competition of our project this year, we strongly recommend that PTRCCS be used by all teams working on related subject. Good news is that there are already commercial products based on this system and hopefully the complete vector DNA containing the system will be soon available. If necessary, please feel free to contact us and we are more than glad to provide the complete vector containing PTRCCS we used this year.

2. Biology

PTRCCS consists of two parts: (1) ParA-ParB-ParC plasmid partition system; (2) oviS/oviV copy-control system.

2.1 ParA-ParB-ParC plasmid partition system：
The ParA-ParB-ParC from the F plasmid in E. coli consists of three elements that are essential for plasmid partition: Protein SopA, Protein SopB and Cis-acting region sopC. The system ensures the proper distribution of newly-replicated plasmids to daughter cells during cell division, when these proteins mutually impact. [4]

2.2 oriS/oriV Copy-Control system：
The oriS/oriV Copy-Control system is comprised of the oriS(ori2)-repE-incC system that controls a single copy of the plasmid and the oriV/(TrfA) system that implements a strictly controllable multicopy. [5] The oriS(ori2)-repE-incC system derives from F plasmid in E. coli, composed of replicon oriS(ori2), protein repE and incompatibility region incC. In the single copy mode, plasmid replication initiates at oriS (ori2), which consists of (1) four directly repeated sequences of 19 bp (iterons), (2) an AT-rich region, and (3) binding sites for the host DnaA protein. The RepE protein (251 residues, 29 kD), when in the monomeric form, mediates the assembly of a replication complex at oriS. The dimeric form of RepE binds to the inverted repeats of the repE operator exerting autogenous repression. [6]
The oriV/(TrfA) system derives from RK2 Vector. The oriV origin of replication consists of eight 17- bp direct repeats (iterons) that bind a monomeric form of the initiation protein TrfA [7]. DNA replication oriV is completely inactive in the commonly used hosts, because they do not produce the TrfA replication protein upon which replication at oriV depends. To supply the TrfA protein, Jadwiga Wild and his partner constructed special hosts, in which the synthesis of copy-up TrfA mutant protein is very tightly controlled by the ParaBAD (PBAD) promoter and AraC protein. [8]

3. Characterization

3.1 PTRCCS verified to be useful
We have not been able to convert the assembled mitochondrial genome of S. cerevisiae into E. coli for stable cloning even after numerous attempts before applying this system. Nonetheless, hardly had we equipped the vectors with this system when we successfully realized the cloning of the assembled mitochondrial genome in Saccharomyces cerevisiae which was later transferred into E. coli for stable cloning.

3.2 Characterization Purpose Jadwiga Wild and his partner pointed out in their work that vectors containing this system were capable of conversion from single copy mode to multiple copy mode only after induction and the copy number was determined by the length of the sequences inserted in the vectors. [8]
Therefore, we measured the copy number of the vector containing the system in the case of inserting the minimal S. cerevisiae genome sequence we designed this year.
We adopted the QPCR method to measure the E. coli plasmid copy number, which was ever carried out by Lee C, Kim J, Shin S G, et al. [9] 

   aljifjaewfjiwajigjiajgilajfijwailfjiwaejfil

3.3 Protocol of Copy Number Determination qPCR
3.3.1 Lysate standard sample qPCR
1.Inoculate a single colony into 5 mL of liquid LB medium with corresponding antibiotic and incubate in the shaker at 37 °C;
2.After 14-16h of growth, transfer 100 μL of suspended cells to 5 mL of fresh liquid LB medium with corresponding antibiotic and incubate at 37 °C until the OD600 reaches 0.7-0.8;
3.Spin down a suspended 1 mL of cells of 0.7 OD600 at 8.0g for 15 min
(Growth conditions are specified at the end of the protocol);
4.Remove the medium and resuspend the cell pellet in 1 mL of PBS;
5.Spin down the suspended of cells at 8.0 g for 15 min;
6.Repeat steps 2 and 3;
7.Completely remove PBS from the cell pellet;
8.Incubate cells at 95 °C for 10 min;
9.Store cells at -20 °C for 10 min;
10.Completely resuspend dry cell pellet in 100 μL of water by pipetting. Then vortex for 30s and spin down;
11.Make an initial dilution by transferring 10 μL of resuspended cell to 40 μL of water. Pipet carefully vortex for 30s and spin down;
12.Make a second dilution by transferring 10 μL of to 90 μL of water. Pipet carefully vortex for 30s and spin down;
13.For X reactions, make two different mixes using chromosome gene and plasmid gene primers:
X*6 μL of water
X*1 μL Forward primer 20 uM
X*1 μL Reverse primer 20 uM
X*10 μL of SYBR Green
14. First, transfer 18 μL of mix with chromosome primers to first X tubes, then transfer 18 μL of plasmid primers mix to other X tubes (X*2 tubes);
15. Add 2 μL of each diluted sample to the tubes;
16. Tenderly close the caps;
17. Run the reaction.