Difference between revisions of "Part:BBa K3137001"

Latest revision as of 20:23, 21 October 2019

Improved RBS 4

This year, we improved the sequence of the RBS BBa_B0033, and got the RBS BBa_K3137001.

Figure1. Maps of the constructed plasmids

As we all know, RBS refers to a sputum-rich untranslated region upstream of the initiation codon AUG. BBa_B0033 is a weaker RBS based on Ron Weiss thesis. Compared with BBa_B0030, BBa_B0031, BBa_B0032, BBa_B0034, BBa_B0033 is the weakest, and whose RBS strength is 0.35% of BBa_B0034. By analyzing their sequence, we found that BBa_B0033 has fewer purines and no not have AAA sequence. SO we designed the RBS by adjusting the proportion of bases.

Figure2. Sequences of the original RBS and improved RBS

Then we ligated the two RBS with promotor rsmH (BBa_K3137000) and gene gfp (BBa_K3137011) and transform into E. coli BL21 to compare their green fluorescence. And take E. coli BL21 as a control. The figure below shows that BBa_K3137001 has the stronger fluorescently protein expression than BBa_B0033, that means it’s efficient to increase the protein expression by increasing the number of purines in the RBS sequence, which is beneficial to the binding of ribosomes.

Figure3. Expression of gfp driven by BBa_K3137001, BBa_B0033 and control

Figure4. Fluorescence intensity of gfp driven by BBa_K3137001, BBa_B0033 and control

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K3137001 short</partinfo>
-AbpAB are two genes of the E.coli genome that express resistant proteins that are resistant to T2,T4,T7 and λ phages. We obtained abpA and abpB by PCR from the genome of E.coli BL21 and constructed recombinant plasmid that connected abpA and abpB at the same time, then used IPTG to induce the expression of resistant protein and verified the resistant function of the protein.
+This year, we improved the sequence of the RBS <bbpart>BBa_B0033</bbpart>, and got the RBS <bbpart>BBa_K3137001</bbpart>.
+[[Image:JiangnanUchina improve 1.png|400px|thumb|center|Figure1. Maps of the constructed plasmids]]
+As we all know, RBS refers to a sputum-rich untranslated region upstream of the initiation codon AUG. <bbpart>BBa_B0033</bbpart> is a weaker RBS based on Ron Weiss thesis. Compared with <bbpart>BBa_B0030</bbpart>, <bbpart>BBa_B0031</bbpart>, <bbpart>BBa_B0032</bbpart>, <bbpart>BBa_B0034</bbpart>, <bbpart>BBa_B0033</bbpart> is the weakest, and whose RBS strength is 0.35% of <bbpart>BBa_B0034</bbpart>. By analyzing their sequence, we found that <bbpart>BBa_B0033</bbpart> has fewer purines and no not have AAA sequence. SO we designed the RBS by adjusting the proportion of bases.
+[[Image:JiangnanUchina improve 2.png|400px|thumb|center|Figure2. Sequences of the original RBS and improved RBS]]
+Then we ligated the two RBS with promotor <i>rsmH</i> (<bbpart>BBa_K3137000</bbpart>) and gene <i>gfp</i> (<bbpart>BBa_K3137011</bbpart>) and transform into <i>E. coli</i> BL21 to compare their green fluorescence. And take <I>E. coli</i> BL21 as a control.
+The figure below shows that BBa_K3137001 has the stronger fluorescently protein expression than <bbpart>BBa_B0033</bbpart>, that means it’s efficient to increase the protein expression by increasing the number of purines in the RBS sequence, which is beneficial to the binding of ribosomes.
+[[Image:JiangnanUchina improve 3.png|400px|thumb|left|Figure3. Expression of <I>gfp</i> driven by <bbpart>BBa_K3137001</bbpart>, <bbpart>BBa_B0033</bbpart> and control]]
+[[Image:JiangnanUchina improve 4.png|400px|thumb|right|Figure4. Fluorescence intensity of <i>gfp</i> driven by <bbpart>BBa_K3137001</bbpart>, <bbpart>BBa_B0033</bbpart> and control]]
+<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>
 <!-- Add more about the biology of this part here