Difference between revisions of "Part:BBa K1949060"
Line 6: Line 6:
 
===Improvement===
 
===Improvement===
  
−
I. Improved Prhl by Tokyo_Tech 2014 and RhlR assay   
+
I. Improved Prhl by Tokyo_Tech 2014 and characterize RhlR assay   
−
We found that Prhl(RL) (BBa_K1529300) strength was weak and the expression level depended on ssrA tag (Fig.1 (a), (b)); ssrA-tagged proteins are prone to be degraded by cellular proteases.  
+
<span style="margin-left: 10px;">We found that Prhl(RL) (BBa_K1529300) activity was weak and the expression level depended on ssrA tag (Fig.1); ssrA-tagged proteins are prone to be degraded by cellular proteases. Prhl(LR) (BBa_K1529310) activity was strong and unexpectedly reacted with C12 (crosstalk) (Fig.4).
  
−
待ち！
+
[[Image:Prhlfig1.png|thumb|center|400px|Fig. 1.  Comparison of the Past improved Prhl  and RhlR]]<br>
−
Fig.1
+
  
−
The colonies of transformants with a rhlR (BBa_ C0171) plasmid looked rough and the growth rate was low(Fig.2left), while the colonies of transformants with a rhlR-ssrA (BBa_C0071) plasmid looked smooth and the growth rate was normal(Fig.2-right). However, the reason for this result is unclear.
+
[[Image:Prhlfig2.png|thumb|center|400px|Fig. 2. Comparison of the Past improved Prhl and RhlR adding high concentration C4]]<br>
  
−
Fig.2
+
<span style="margin-left: 10px;">The colonies of transformants with a rhlR (BBa_C0171) plasmid looked rough and the growth rate was low(Fig.2-left), while the colonies of transformants with a rhlR-ssrA (BBa_C0071) plasmid looked smooth and the growth rate was normal(Fig.2-right). However, the reason for this result is unclear.
 +
 
 +
[[Image:Prhlfig3.png|thumb|center|400px|Fig. 3.  The colonies of transformants with a rhlR (left) or a rhlR-ssrA（right)]]<br>
  
 
II. Improvement the native Prhl  
 
II. Improvement the native Prhl  
−
By introducing a single point mutation into native Prhl (BBa_R0071) by PCR, we obtained 198 Prhl mutants and chose the two Prhl mutants of which promoter strength was stronger than native Prhl (Fig.3　※この図は差し替えます).
+
<span style="margin-left: 10px;">By introducing a single point mutation into native Prhl (BBa_R0071) by PCR, we obtained 198 Prhl mutants and chose the two Prhl mutants of which promoter activity was stronger than native Prhl(Fig.4).
−
The sequences of these tow chosen mutants are shown in below. The small characters indicate the scar sequence and a single point mutation is colored with red.
+
−
 
+
−
Native Prhl (BBa_R0071)
+
−
TCCTGTGAAATCTGGCAGTTACCGTTAGCTTTCGAATTGGCTAAAAAGTGTTCtactagagAAAGAGGAGAAA
+
−
 
+
−
Prhl(D6) (BBa_K1949060)
+
−
TCCTGTGAAATCTGGCAGTTACCGTTAGCTTTCGAATTGGCTAtAAAGTGTTCtactagagAAAGAGGAGAAA
+
−
+
−
Prhl(H7) TCCTGTGAAATCTGGCAGTTACCGTTAGCTTTCGAATTGGCTAtAAAGTGTTCtactagtaAAAGAGGAGAAA   
+
−
 
+
−
Fig.3             
+
  
 +
[[Image:Prhlfig4.png|thumb|center|400px|Fig. 4.  RFU of GFP / turbidity of imoroved Prhl mutants]]<br>
  
 
III. Comparison the improved Prhl by Tokyo_Tech 2014 to our original improved Prhl
 
III. Comparison the improved Prhl by Tokyo_Tech 2014 to our original improved Prhl
 +
<span style="margin-left: 10px;">Prhl(NM) was chosen from the many Prhl mutants, and by comparing Prhl(NM) to Prhl(LR), we obtained the result below (Fig.3-2-2-3-4). The reaction activity of Prhl(NM) to C4 was stronger than that of Prhl(LR), and Prhl(NM) did not react with C12 at all.
  
−
Prhl(D6) was chosen from the many Prhl mutants, and by comparing Prhl(D6) to Prhl(LR), we obtained the result below (Fig.3-2-2-3-4). The reaction strength of Prhl(D6) to C4 was higher than that of Prhl(LR), and Prhl(D6) did not react with C12 at all.
+
[[Image:Prhlfig5.png|thumb|center|400px|Fig. 5. Comparison of Prhl(NM) to Prhl(LR)]]<br>
−
 
+
−
Fig.4
+

Revision as of 07:26, 13 October 2016

Prhl(NM)-rbs-gfp

We simulated our final genetic circuits and found that the circuits did not work, because Prhl strength was too weak. (see our wikiリンク貼る). We therefore considered using the improved Prhl (BBa_K1529310, BBa_K1529300) established by Tokyo_Tech 2014, but we noticed that they were inappropriate for two reasons (see our wiki　). Then, we decided to improve Prhl Promoter and obtain our original improved Prhl (BBa_K1949060) that suited our goal.

Improvement

I. Improved Prhl by Tokyo_Tech 2014 and characterize RhlR assay We found that Prhl(RL) (BBa_K1529300) activity was weak and the expression level depended on ssrA tag (Fig.1); ssrA-tagged proteins are prone to be degraded by cellular proteases. Prhl(LR) (BBa_K1529310) activity was strong and unexpectedly reacted with C12 (crosstalk) (Fig.4).

Fig. 1. Comparison of the Past improved Prhl and RhlR

Fig. 2. Comparison of the Past improved Prhl and RhlR adding high concentration C4

The colonies of transformants with a rhlR (BBa_C0171) plasmid looked rough and the growth rate was low(Fig.2-left), while the colonies of transformants with a rhlR-ssrA (BBa_C0071) plasmid looked smooth and the growth rate was normal(Fig.2-right). However, the reason for this result is unclear.

Fig. 3. The colonies of transformants with a rhlR (left) or a rhlR-ssrA（right)

II. Improvement the native Prhl By introducing a single point mutation into native Prhl (BBa_R0071) by PCR, we obtained 198 Prhl mutants and chose the two Prhl mutants of which promoter activity was stronger than native Prhl(Fig.4).

Fig. 4. RFU of GFP / turbidity of imoroved Prhl mutants

III. Comparison the improved Prhl by Tokyo_Tech 2014 to our original improved Prhl Prhl(NM) was chosen from the many Prhl mutants, and by comparing Prhl(NM) to Prhl(LR), we obtained the result below (Fig.3-2-2-3-4). The reaction activity of Prhl(NM) to C4 was stronger than that of Prhl(LR), and Prhl(NM) did not react with C12 at all.

Fig. 5. Comparison of Prhl(NM) to Prhl(LR)