Difference between revisions of "Part:BBa K1017404"
(→‎sRNA Inhibit Efficiency)
 
(26 intermediate revisions by 6 users not shown)
Line 2: Line 2:
 
<partinfo>BBa_K1017404 short</partinfo>
 
<partinfo>BBa_K1017404 short</partinfo>
  
−
A DNA sequence whose product is a small RNA, which can bind on the complement sequence of itself.
+
===Description of function===
 +
<p>Small RNA is an important role on gene regulation.
 +
It will interact with the targeted mRNA by imperfect basepairing,
 +
reducing the translational efficiency and recruiting chaperones such as
 +
Hfq for translational termination.Since we choose sRNA to be part of our regulated-system, we need to prevent
 +
the sRNA from effecting undesired genes, so it is necessarily to design
 +
artificial sRNA that targets specifically to the desired genes.</p>
  
−
The sRNA will base pair to target mRNA,and lower the translation efficiency. The sRNA bind on mRNA will turn into a shape like hairpin, and recruit chaperones such as Hfq to prevent the sRNA-mRNA combination from degrading.
+
<p>The following figure depicts the mechnism of sRNA. The sRNA basepair with
 +
the target mRNA and the ribosome can't bind the mRNA. Therefore, the translation
 +
would be inhibited.</p>
 +
[[File:Nctu_formosa_sRNA_inhibition.JPG|thumb|500px|center|Figure 1. '''sRNA-Hfq complex specifically binds on target gene, forming a blockade of ribosome binding. Therefore, the translation is inhibited.''']]
  
−
[[File:NCTU sRNA-2 structure predict.png]]
+
===How we design===
 +
We first picked the sRNA from a library of artificial sRNA that was constructed by fusing a randomized antisense domain of Spot42, the scaffold that is known to recruit the RNA chaperons. As we want the sRNA we picked can specifically complementary to the SD sequence which in our designed RBS(BBa_K1017202[https://parts.igem.org/wiki/index.php?title=Part:BBa_K1017202]), we picked the one which contains a consensus sequence, 5’-CCCUC-3’. Our artificial sRNA also has three stem-loop double stranded RNA structures, and the loop closest to the 3’ terminus is complementary to a sequence preceding the initiation codon of mRNA, so that it can prevent the ribosome from binding to the initiation condon, so the translation would be repressed.
  
 +
[[File:NCTU sRNA-2 structure predict.png|thumb|600px|center|Figure 2. '''sRNA-Hfq complex specifically binds on target gene, forming a blockade of ribosome binding. Therefore, the translation is inhibited.''']]
 +
 +
===Effect on E. Coli Growth===
 +
To test whether or not our sRNA would effect the growth of E. Coli, we compared the growth of E. Coli with PSB1C3 (without RFP) and the E. Coli growth with Pcons + sRNA. It can be observed from the following figure that the resultant growth curves are similar, showing no signs of growth interference. This result proves that our sRNA regulated system can be integrated into bacteria.
 +
 +
[[File:NCTU_sRNA_growth.JPG|thumb|600px|center|Figure 3.'''The growth curve of the E. Coli with sRNA shows uninterrupted growth curve that is similar to the growth curve of the E. Coli with PSB1C3.''']]
 +
 +
Then, We employed the following biobricks to test the regulation efficiency of the sRNA we designed :
 +
 +
[[File:NCTU_sRNA_regulation_biobrick.png|thumb|600px|center|Figure 4.'''The biobrick of testing the sRNA.''']]
 +
 +
From the following figure, it can be observed that sRNA can regulate RFP expression. The bacterial colonies without sRNA are red, showing high RFP expression. The bacterial colonies with sRNA, however, are white, suggesting that sRNA has regulated the RFP expression by base pairing to rRBS. The normalized expression without sRNA must be much higher than the expression with sRNA, or else we would not be able to easily distinguish the difference in RFP expression through the colors of the colonies.
 +
 +
[[File:NCTU_sRNA_regulation_plate.JPG|thumb|600px|center|Figure 5.'''The bacterial colonies with sRNA shows no clear sign of RFP expression, while the colonies without sRNA do.''']]
 +
 +
===sRNA Inhibit Efficiency===
 +
<p>We co-transformed the following circuits into E.coli to test the efficiency of sRNA:</p>
 +
 +
<p>'''Pcons + rRBS + mRFP + Ter''' </p>
 +
 +
<p>'''Pcons + sRNA'''</p>
 +
Figure 6 can be observed that sRNA can regulate RFP expression. The bacterial colonies without sRNA(green curve) expressed fluorescence higher than the colonies with sRNA(blue curve). This proved that sRNA we designed really had its repressive function.
 +
 +
[[file:NCTU_result_sRNA_Inhibit_Efficiency.png|thumb|600px|center|Figure 6.'''Colony A shows sRNA regulation with high efficiency.''']]
 +
 +
===Acknowedgment of sources and references===
 +
Torsten Waldminghaus, Nadja Heidrich, Sabine Brantl and Franz Narberhaus .(2012). Engineering Artificial Small RNAs for Conditional Gene Silencing in Escherichia coli ,1: 6–13
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Latest revision as of 11:33, 3 November 2013

sRNA-2

Description of function

Small RNA is an important role on gene regulation. It will interact with the targeted mRNA by imperfect basepairing, reducing the translational efficiency and recruiting chaperones such as Hfq for translational termination.Since we choose sRNA to be part of our regulated-system, we need to prevent the sRNA from effecting undesired genes, so it is necessarily to design artificial sRNA that targets specifically to the desired genes.

The following figure depicts the mechnism of sRNA. The sRNA basepair with the target mRNA and the ribosome can't bind the mRNA. Therefore, the translation would be inhibited.

Figure 1. sRNA-Hfq complex specifically binds on target gene, forming a blockade of ribosome binding. Therefore, the translation is inhibited.

How we design

We first picked the sRNA from a library of artificial sRNA that was constructed by fusing a randomized antisense domain of Spot42, the scaffold that is known to recruit the RNA chaperons. As we want the sRNA we picked can specifically complementary to the SD sequence which in our designed RBS(BBa_K1017202[1]), we picked the one which contains a consensus sequence, 5’-CCCUC-3’. Our artificial sRNA also has three stem-loop double stranded RNA structures, and the loop closest to the 3’ terminus is complementary to a sequence preceding the initiation codon of mRNA, so that it can prevent the ribosome from binding to the initiation condon, so the translation would be repressed.

Figure 2. sRNA-Hfq complex specifically binds on target gene, forming a blockade of ribosome binding. Therefore, the translation is inhibited.

Effect on E. Coli Growth

To test whether or not our sRNA would effect the growth of E. Coli, we compared the growth of E. Coli with PSB1C3 (without RFP) and the E. Coli growth with Pcons + sRNA. It can be observed from the following figure that the resultant growth curves are similar, showing no signs of growth interference. This result proves that our sRNA regulated system can be integrated into bacteria.

Figure 3.The growth curve of the E. Coli with sRNA shows uninterrupted growth curve that is similar to the growth curve of the E. Coli with PSB1C3.

Then, We employed the following biobricks to test the regulation efficiency of the sRNA we designed :

Figure 4.The biobrick of testing the sRNA.

From the following figure, it can be observed that sRNA can regulate RFP expression. The bacterial colonies without sRNA are red, showing high RFP expression. The bacterial colonies with sRNA, however, are white, suggesting that sRNA has regulated the RFP expression by base pairing to rRBS. The normalized expression without sRNA must be much higher than the expression with sRNA, or else we would not be able to easily distinguish the difference in RFP expression through the colors of the colonies.

Figure 5.The bacterial colonies with sRNA shows no clear sign of RFP expression, while the colonies without sRNA do.

sRNA Inhibit Efficiency

We co-transformed the following circuits into E.coli to test the efficiency of sRNA:

Pcons + rRBS + mRFP + Ter

Pcons + sRNA

Figure 6 can be observed that sRNA can regulate RFP expression. The bacterial colonies without sRNA(green curve) expressed fluorescence higher than the colonies with sRNA(blue curve). This proved that sRNA we designed really had its repressive function.

Figure 6.Colony A shows sRNA regulation with high efficiency.

Acknowedgment of sources and references

Torsten Waldminghaus, Nadja Heidrich, Sabine Brantl and Franz Narberhaus .(2012). Engineering Artificial Small RNAs for Conditional Gene Silencing in Escherichia coli ,1: 6–13 Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 3
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]