Difference between revisions of "Part:BBa K3490030"

Latest revision as of 13:24, 26 October 2020

The toxin-antitoxin system : hicA-hicB

The final version of growth switch

During the experiment process, we had difficulty constructing both plasmid. With the doubt that the EL222-FLP system might be harmful to the growth of bacteria, we changed the design of blue-sensitive protein EL222 into thermo-sensitive protein CI857 on the plasmid pCP20 provided by our PI, Prof. Ng (Fig.4).

Fig.1. The plasmid map which changes the toxin-antitoxin gene into fluorescent genes

Fig.2. The heat-activated plasmid pCP20

The thermo-sensitive system: pCP20

Therefore, we employed heat-activation, as our new method to resuscitate the bacteria. The thermo-sensitive system can be activated through the degradation of CI857 protein when the temperature rises to 36-40 ºC.^[1] This time, the experiments of ligation and transformation were successful, but the color of GFP were not observed in the bacteria after transformation. Suspecting that the sequence might be mutated, we sent plasmid for DNA sequencing. As we expected, the sequencing region matched poorly to our GFP-OFP design. As shown in Fig. 4, one of the FRT sequences is severely mutated next to the promoter J23100(Fig.5). This suggests that our design had violated some fundamental principle. Since the FLP recombinase works by twisting the DNA to bring two FRT sites together and nicking them to generate insertion or deletion. We speculated that the distance between the two FRT sites is insufficient for the two FRT sites to form a loop in vivo. We then check for the optimal distance between two FRT sites for the FRT-FLP system to function. Previous report states that the optimal distance for the recombination to take place is around 200bp in vitro^[2], while our original design between two FRT sites is only 160bp, this may be the cause of malfunction.
Nevertheless, we had to come up with a new design.

Fig.3. The sequencing result of sfGFP-FRT-J23100-FRT-OFP, J23100 is highlighted, and the red arrow indicates FRT sequence flanking J23100

The change of FRT mechanism

FRT sites can either change the orientation of the gene or delete the gene depending on the orientation of the two FRT sequences^[3]. Since the design of changing the promoter direction failed, we choose to delete the gene, which is more common in general lab settings. For the new design, we still used the plasmid pCP20 for heat-activation, but we put hicA gene between the FRT sites for deletion on the plasmid pSB1C3 (Fig 6.a), and the plasmid pSB3K3 with hicB gene was also designed to continuously produce HicB (Fig 6.b). Because pSB3K3 is a low copy number plasmid, the expression of HicB is less than HicA, therefore, the bacteria will hibernate. After heat activation, hicA gene will be deleted while hicB gene is still expressed, so the bacteria can resuscitate. For these three plasmids, we can regulate the growth of bacteria, meeting our need to design a growth switch.

Fig.4. The design of BBa_K3490030

Fig.5. The design of BBa_K3490031

The experiment of kanamycin-resistant gene deletion: Prove of design

Due to time constraint, we can only demonstrate this model using pKD3 plasmid, which carries a kanamycin-resistance gene flanked between two FRT sites provided by our PI, Prof. Ng. If the bacteria cannot survive at LB plate with kanamycin after heat-activation, we can demonstrate that the gene between the FRT sites was deleted, indicating that our design is feasible. For the experiment, we co-transformed pKD3 and pCP20 into E. coli DH5α, and cultured a colony in 37℃ for 8 hr and transfer to 42℃ for 1 hr to heat-activate the bacteria, and then spread on the LB plate (dilute 105 times) and culture for 12 hr. After the colony grew, we screened the colony on a kanamycin plate to check whether the bacteria will grow or not. Results show that most of the bacteria can not survive on the Kanamycin plate but can still survive on the LB plate without kanamycin (Fig.7, 8.), which means the antibiotic-resistance gene was deleted successfully. With this data, we are convinced that the design of pSB3K3 with hicA gene is feasible.

Fig.6. The bacteria can grow on the LB plate without antibiotics (the left one), but when we pick the colony from it to the kanamycin plate (the right one), there is almost no colony on the plate.

Lane1: the uncut plasmid with activation of the FRT system

Lane2: the plasmid cut by HindIII with activation of the FRT system

Lane3: the uncut plasmid without activation of the FRT system

Lane4: the plasmid cut by HindII without activation of the FRT system

LaneM: the marker

Fig.7. The plasmid pattern before and after activating FRT genes checked by DNA gel electrophoresis.

Result: Predicting success

Reference

[1]Jechlinger W, Szostak MP, Witte A, Lubitz W. Altered temperature induction sensitivity of the lambda pR/cI857 system for controlled gene expression in Escherichia coli. FEMS Microbiology Letters. 1999;173(2):347-352.
[2]Ringrose L. Quantitative comparison of DNA looping in vitro and in vivo: chromatin increases effective DNA flexibility at short distances. The EMBO Journal. 1999;18(23):6630-6641.
[3]Park Y-N, Masison D, Eisenberg E, Greene LE. Application of the FLP/FRT system for conditional gene deletion in yeast Saccharomyces cerevisiae. Yeast. 2011;28(9):673-681.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 1115
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 1055
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K3490030 short</partinfo>
-<br><b style="font-size:1.5rem">Overview</b>
+<br><b style="font-size:1.0rem">The final version of growth switch</b>
-<br>For our project, we need the bacteria to stay alive in our contact lenses until it can be used. However, bacteria can’t live for a long time in such a small space, especially with such limited nutrients. To solve this problem, we designed a growth switch to put bacteria into hibernation for storage, which we can then resuscitate after exposure to external stimulation.
-<html>
 <br>
-<div style="width=100%; display:flex; align-items: center; justify-content: center;">
+<br>During the experiment process, we had difficulty constructing both plasmid. With the doubt that the EL222-FLP system might be harmful to the growth of bacteria, we changed the design of blue-sensitive protein EL222 into thermo-sensitive protein CI857 on the plasmid pCP20 provided by our PI, Prof. Ng (Fig.4).
-<img src="https://static.igem.org/mediawiki/parts/4/42/T--NCKU_Tainan--BBa_K3490020.gif" style="width:35%;">
-</div>
-<br>Fig.1. The big picture of growth switch
-</html>
-<br><b style="font-size:1.5rem">Toxin-antitoxin system TA System</b>
-The <i>hicA</i>-<i>hicB</i> locus located on <i>E. coli</i> chromosome is a toxin-antitoxin system (TA system). The TA system is composed of linked genes, encoding a toxic protein that can inhibit cell growth and an antitoxic protein which neutralize the toxic protein<sup>[1]</sup>.  For our project, a kind of the TA system effect called the stress tolerance effect, is used to induce bacteria hibernation. By making use of this mechanism of the TA system, we designed a growth switch to regulate the growth of bacteria.
-According to a research<sup>[2]</sup>, HicA is a toxin promoting mRNA degradation, leading to the hibernation of bacteria, while HicB is an antitoxin that can neutralize the effect of HicA. Hence, through regulating the expression of hicA and hicB genes, we can control the growth of bacteria, hibernating the bacteria before the contact lens are used.
-<br><b style="font-size:1.5rem">The blue light-sensitive system: EL222</b>
-Since contact lens are sealed during the process of production, it is impossible to regulate the growth switch through chemical induction; hence, we turned to external stimuli such as light, temperature, etc. At first, we used EL222 to regulate the system<sup>[3]</sup> (a blue light-sensitive protein), <i>pBlind</i> (a promoter activated by EL222), FLP, and <i>FRT</i> to change the orientation of our promoter<sup>[4]</sup>, which is activated by blue light (Fig.2). In other words, we have to illuminate the contact lens with blue light for 30 minutes before use.
-<html>
-<br>
-<div style="width=100%; display:flex; align-items: center; justify-content: center;">
-<img src="https://static.igem.org/mediawiki/parts/7/7b/T--NCKU_Tainan--BBa_K3490020-ver2.png" style="width:35%;">
-</div>
-<br>Fig.2. The first version of design: BBa_K3490020. This design enables us to control the bacteria growth in three stages.
-</html>
-<br>First, the production stage is when we are culturing our bacteria. Without any arabinose addition during culture, the bacteria will be able to grow normally. After the production process, we will put the bacteria and arabinose into the contact lens together, so the <i>pBAD</i> promoter will transcribe <i>hicA</i>, causing the bacteria to hibernate. When we need to resuscitate the bacteria, <i>EL222</i> will be activated by blue light to induce <i>pBlind</i> promoter, and then FLP will be transcribed. FLP will act on the <i>FRT</i> sites to change the orientation of the <i>pBAD</i> promoter, as a result, the bacteria will start to transcribe hicB to neutralize HicA and thus continue to grow.
-<br>We replaced <i>hicA</i> and <i>hicB</i> with <i>GFP</i> and <i>OFP</i> gene to build a test plasmid because it is much easier to observe the color change than to count the CFU (Fig.3). If the color of fluorescence changes from green to orange, the experiment is successful to prove that the design is feasible.
-<br>However, during the experiment process, we had difficulty constructing both plasmid. With the doubt that the EL222-FLP system might be harmful to the growth of bacteria, we changed the design of blue-sensitive protein EL222 into thermo-sensitive protein CI857 on the plasmid pCP20 provided by our PI, Prof. Ng (Fig.4).
 <html>
 <br>
@@ Line 39: / Line 11: @@
 <img src="https://static.igem.org/mediawiki/parts/e/ed/T--NCKU_Tainan--BBa_K3490021-ver2.png" style="width:35%;">
 </div>
-<br>Fig.3. The plasmid map which changes the toxin-antitoxin gene into fluorescent genes
+<p align="center">Fig.1. The plasmid map which changes the toxin-antitoxin gene into fluorescent genes</p>
 </html>
 <html>
@@ Line 46: / Line 18: @@
 <img src="https://static.igem.org/mediawiki/parts/f/f7/PCP20.png" style="width:35%;">
 </div>
-<br>Fig.4. The heat-activated plasmid pCP20
+<p align="center">Fig.2. The heat-activated plasmid pCP20</p>
 </html>
 <br>
 <br><b style="font-size:1.5rem">The thermo-sensitive system: pCP20</b>
-<br>Therefore, we employed heat-activation, as our new method to resuscitate the bacteria. The thermo-sensitive system can be activated through the degradation of CI857 protein when the temperature rises to 36-40 ºC.<sup>[5]</sup> This time, the experiments of ligation and transformation were successful, but the color of GFP were not observed in the bacteria after transformation. Suspecting that the sequence might be mutated, we sent plasmid for DNA sequencing. As we expected, the sequencing region matched poorly to our GFP-OFP design. As shown in Fig. 4, one of the <i>FRT</i> sequences is severely mutated next to the promoter <i>J23100</i>(Fig.5). This suggests that our design had violated some fundamental principle.
+<br>
-Since the FLP recombinase works by twisting the DNA to bring two <i>FRT</i> sites together and nicking them to generate insertion or deletion. We speculated that the distance between the two <i>FRT</i> sites is insufficient for the two <i>FRT</i> sites to form a loop in vivo. We then check for the optimal distance between two <i>FRT</i> sites for the <i>FRT</i>-FLP system to function.  Previous report states that the optimal distance for the recombination to take place is around 200bp in vitro<sup>[6]</sup>, while our original design between two <i>FRT</i> sites is only 160bp, this may be the cause of malfunction.
+<br>Therefore, we employed heat-activation, as our new method to resuscitate the bacteria. The thermo-sensitive system can be activated through the degradation of CI857 protein when the temperature rises to 36-40 ºC.<sup>[1]</sup> This time, the experiments of ligation and transformation were successful, but the color of GFP were not observed in the bacteria after transformation. Suspecting that the sequence might be mutated, we sent plasmid for DNA sequencing. As we expected, the sequencing region matched poorly to our GFP-OFP design. As shown in Fig. 4, one of the <i>FRT</i> sequences is severely mutated next to the promoter <i>J23100</i>(Fig.5). This suggests that our design had violated some fundamental principle.
+Since the FLP recombinase works by twisting the DNA to bring two <i>FRT</i> sites together and nicking them to generate insertion or deletion. We speculated that the distance between the two <i>FRT</i> sites is insufficient for the two <i>FRT</i> sites to form a loop in vivo. We then check for the optimal distance between two <i>FRT</i> sites for the <i>FRT</i>-FLP system to function.  Previous report states that the optimal distance for the recombination to take place is around 200bp in vitro<sup>[2]</sup>, while our original design between two <i>FRT</i> sites is only 160bp, this may be the cause of malfunction.
 <br>Nevertheless, we had to come up with a new design.
@@ Line 57: / Line 30: @@
 <br>
 <div style="width=100%; display:flex; align-items: center; justify-content: center;">
-<img src="https://static.igem.org/mediawiki/parts/5/55/T--NCKU_Tainan--BBa_K3490020-seq.png" style="width:35%;">
+<img src="https://static.igem.org/mediawiki/parts/5/55/T--NCKU_Tainan--BBa_K3490020-seq.png" style="width:45%;">
 </div>
-<br>Fig.5. The sequencing result of <i>sfGFP</i>-<i>FRT</i>-<i>J23100</i>-<i>FRT</i>-<i>OFP</i>, <i>J23100</i> is highlighted, and the red arrow indicates <i>FRT</i> sequence flanking <i>J23100</i>
+<p align="center">Fig.3. The sequencing result of <i>sfGFP</i>-<i>FRT</i>-<i>J23100</i>-<i>FRT</i>-<i>OFP</i>, <i>J23100</i> is highlighted, and the red arrow indicates <i>FRT</i> sequence flanking <i>J23100</i></p>
 </html>
 <br>
 <br><b style="font-size:1.5rem">The change of <i>FRT mechanism</i></b>
-<br><i>FRT</i> sites can either change the orientation of the gene or delete the gene depending on the orientation of the two <i>FRT</i> sequences<sup>[7]</sup>. Since the design of changing the promoter direction failed, we choose to delete the gene, which is more common in general lab settings. For the new design, we still used the plasmid pCP20 for heat-activation, but we put <i>hicA</i> gene between the <i>FRT</i> sites for deletion on the plasmid pSB1C3 (Fig 6.a), and the plasmid pSB3K3 with <i>hicB</i> gene was also designed to continuously produce HicB (Fig 6.b).  Because pSB3K3 is a low copy number plasmid, the expression of HicB is less than HicA, therefore, the bacteria will hibernate. After heat activation, hicA gene will be deleted while hicB gene is still expressed, so the bacteria can resuscitate. For these three plasmids, we can regulate the growth of bacteria, meeting our need to design a growth switch.
+<br>
+<br><i>FRT</i> sites can either change the orientation of the gene or delete the gene depending on the orientation of the two <i>FRT</i> sequences<sup>[3]</sup>. Since the design of changing the promoter direction failed, we choose to delete the gene, which is more common in general lab settings. For the new design, we still used the plasmid pCP20 for heat-activation, but we put <i>hicA</i> gene between the <i>FRT</i> sites for deletion on the plasmid pSB1C3 (Fig 6.a), and the plasmid pSB3K3 with <i>hicB</i> gene was also designed to continuously produce HicB (Fig 6.b).  Because pSB3K3 is a low copy number plasmid, the expression of HicB is less than HicA, therefore, the bacteria will hibernate. After heat activation, hicA gene will be deleted while hicB gene is still expressed, so the bacteria can resuscitate. For these three plasmids, we can regulate the growth of bacteria, meeting our need to design a growth switch.
 <html>
 <br>
@@ Line 69: / Line 43: @@
 <img src="https://static.igem.org/mediawiki/parts/7/7a/T--NCKU_Tainan--BBa_K3490030-ver2.png" style="width:35%;">
 </div>
-<br>Fig.6.a. The design of BBa_K3490030
+<p align="center">Fig.4. The design of BBa_K3490030</p>
 </html>
 <html>
@@ Line 76: / Line 50: @@
 <img src="https://static.igem.org/mediawiki/parts/b/b1/T--NCKU_Tainan--BBa_K3490031.png" style="width:35%;">
 </div>
-<br>Fig.6.b. The design of BBa_K3490031
+  <p align="center">Fig.5. The design of BBa_K3490031</p>
 </html>
 <br>
 <br><b style="font-size:1.5rem">The experiment of kanamycin-resistant gene deletion: Prove of design</b>
+<br>
 <br>Due to time constraint, we can only demonstrate this model using pKD3 plasmid, which carries a kanamycin-resistance gene flanked between two <i>FRT</i> sites provided by our PI, Prof. Ng. If the bacteria cannot survive at LB plate with kanamycin after heat-activation, we can demonstrate that the gene between the <i>FRT</i> sites was deleted, indicating that our design is feasible. For the experiment, we co-transformed pKD3 and pCP20 into <i>E. coli</i> DH5α, and cultured a colony in 37℃ for 8 hr and transfer to 42℃ for 1 hr to heat-activate the bacteria, and then spread on the LB plate (dilute 105 times) and culture for 12 hr. After the colony grew, we screened the colony on a kanamycin plate to check whether the bacteria will grow or not. Results show that most of the bacteria can not survive on the Kanamycin plate but can still survive on the LB plate without kanamycin (Fig.7, 8.), which means the antibiotic-resistance gene was deleted successfully. With this data, we are convinced that the design of pSB3K3 with <i>hicA</i> gene is feasible.
 <html>
@@ Line 86: / Line 61: @@
 <img src="https://static.igem.org/mediawiki/parts/5/54/Plate_img.png" style="width:35%;">
 </div>
-<br>Fig.7.  The bacteria can grow on the LB plate without antibiotics (the left one), but when we pick the colony from it to the kanamycin plate (the right one), there is almost no colony on the plate.
+<p align="center">Fig.6.  The bacteria can grow on the LB plate without antibiotics (the left one), but when we pick the colony from it to the kanamycin plate (the right one), there is almost no colony on the plate.</p>
 </html>
 <html>
@@ Line 93: / Line 68: @@
 <img src="https://static.igem.org/mediawiki/parts/0/06/T--NCKU_Tainan--BBa_K3490030-gel_ver3.png" style="width:35%;">
 </div>
-<br>Lane1: the uncut plasmid with activation of the <i>FRT</i> system
+<p align="center">Lane1: the uncut plasmid with activation of the <i>FRT</i> system</p>
-<br>Lane2: the plasmid cut by HindIII with activation of the <i>FRT</i> system
+<p align="center">Lane2: the plasmid cut by HindIII with activation of the <i>FRT</i> system</p>
-<br>Lane3: the uncut plasmid without activation of the <i>FRT</i> system
+<p align="center">Lane3: the uncut plasmid without activation of the <i>FRT</i> system</p>
-<br>Lane4: the plasmid cut by HindII without activation of the <i>FRT</i> system
+<p align="center">Lane4: the plasmid cut by HindII without activation of the <i>FRT</i> system</p>
-<br>LaneM: the marker
+<p align="center">LaneM: the marker</p>
 </html>
 <html>
@@ Line 104: / Line 79: @@
 <img src="https://static.igem.org/mediawiki/parts/1/12/T--NCKU_Tainan--BBa_K3490031_flp_function.png" style="width:35%;">
 </div>
-<br>Fig.8. The plasmid pattern before and after activating <i>FRT</i> genes checked by DNA gel electrophoresis.
+<p align="center">Fig.7. The plasmid pattern before and after activating <i>FRT</i> genes checked by DNA gel electrophoresis.</p>
 </html>
+<br>
+<br><b style="font-size:1.0rem">Result: Predicting success </b>
+<br>
+<br>
 <br><b style="font-size:1.5rem">Reference</b>
-<br>[1]Guglielmini J, Van Melderen L. Bacterial toxin-antitoxin systems. Mobile Genetic Elements. 2011;1(4):283-306.
+<br>
-<br>[2]Jørgensen MG, Pandey DP, Jaskolska M, Gerdes K. HicA of Escherichia coli Defines a Novel Family of Translation-Independent mRNA Interferases in Bacteria and Archaea. Journal of Bacteriology. 2008;191(4):1191-1199.‌
+<br>[1]Jechlinger W, Szostak MP, Witte A, Lubitz W. Altered temperature induction sensitivity of the lambda pR/cI857 system for controlled gene expression in Escherichia coli. FEMS Microbiology Letters. 1999;173(2):347-352.
-<br>[3]Motta-Mena LB, Reade A, Mallory MJ, et al. An optogenetic gene expression system with rapid activation and deactivation kinetics. Nature Chemical Biology. 2014;10(3):196-202.
+<br>[2]Ringrose L. Quantitative comparison of DNA looping in vitro and in vivo: chromatin increases effective DNA flexibility at short distances. The EMBO Journal. 1999;18(23):6630-6641.
-<br>[4]Branda CS, Dymecki SM. Talking about a Revolution. Developmental Cell. 2004;6(1):7-28.
+<br>[3]Park Y-N, Masison D, Eisenberg E, Greene LE. Application of the FLP/FRT system for conditional gene deletion in yeast Saccharomyces cerevisiae. Yeast. 2011;28(9):673-681.
-<br>[5]Jechlinger W, Szostak MP, Witte A, Lubitz W. Altered temperature induction sensitivity of the lambda pR/cI857 system for controlled gene expression in Escherichia coli. FEMS Microbiology Letters. 1999;173(2):347-352.
-<br>[6]Ringrose L. Quantitative comparison of DNA looping in vitro and in vivo: chromatin increases effective DNA flexibility at short distances. The EMBO Journal. 1999;18(23):6630-6641.
-<br>[7]Park Y-N, Masison D, Eisenberg E, Greene LE. Application of the FLP/FRT system for conditional gene deletion in yeast Saccharomyces cerevisiae. Yeast. 2011;28(9):673-681.