Difference between revisions of "Part:BBa K2996700"

Latest revision as of 23:51, 21 October 2019

Flexible Linker 4 that separates RpoA and dCas9

FL4 contains 5 repeats of GGGGS, which is a common design of flexible protein structures. Unfortunately, highly-repeating sequence is difficult to deal with during molecular cloning. We have tried these two DNA sequences for elongated FL4 (eFL4), however overlap extension PCR during these two trials both end up in failure.
>eFL4 CTGAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGG CGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCG GCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGC GGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGG CGGCGGCAGCGCGGCGGCG
>eFL4 Optimized by GeneScript TTATCTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTAGTGGTGGTGG TGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTAGCGGTGGTGGTGGTTCTGGTGGTG GTGGTAGTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCAGGTGGTGGTGGTTCGGGTGGT GGTGGTTCTGGTGGTGGTGGTAGTGGTGGTGGTGGTTCGGGTGGTGGTGGTTCTGGTGG TGGTGGTTCAGCAGCAGCA

So, we optimized FL4 sequence ourselves, with a simple Python program that randomly select codons for FL4: CTGTCCGGAGGAGGCGGGTCCGGCGGGGGTGGCTCCGGAGGCGGGGGATCGGGTGGAGGGGGTTCTGCTGCCGCG

Results

Figure 1. Overlap Extension PCR for rpoA-FL4-dCas9

Based on this, we constructed plasmid pActivator without sgRNA and pActivator with sgRNA.

Usage

Using overlap PCR, RpoA is linked to dcas9 through a modified flexible Linker 4 (FL4), which has been proved to be effectively separate bifunction proteins. Now we got the transcription activator BBa_K2996701.

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 5: / Line 5: @@
 FL4 contains 5 repeats of GGGGS, which is a common design of flexible protein structures.
 Unfortunately, highly-repeating sequence is difficult to deal with during molecular cloning. We have tried these two DNA sequences for elongated FL4 (eFL4), however overlap extension PCR during these two trials both end up in failure.
->eFL4
+<br> >eFL4
 CTGAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGG CGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCG GCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGC GGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGG CGGCGGCAGCGCGGCGGCG
->eFL4 Optimized by GeneScript
+<br> >eFL4 Optimized by GeneScript
 TTATCTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTAGTGGTGGTGG TGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTAGCGGTGGTGGTGGTTCTGGTGGTG GTGGTAGTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCAGGTGGTGGTGGTTCGGGTGGT GGTGGTTCTGGTGGTGGTGGTAGTGGTGGTGGTGGTTCGGGTGGTGGTGGTTCTGGTGG TGGTGGTTCAGCAGCAGCA
@@ Line 14: / Line 14: @@
 ===Results===
+<center>{{#tag:html|<img style="max-width: 50%" src="https://static.igem.org/mediawiki/parts/a/ae/T--SJTU-BioX-Shanghai--result_gel1.png" alt="" />}}</center>
+<center><b>Figure 1.</b> <i> Overlap Extension PCR for rpoA-FL4-dCas9</i> </center>
-Figure 2.5. Overlap Extension PCR for rpoA-FL4-dCas9.
+<p> Based on this, we constructed plasmid pActivator without sgRNA and pActivator with sgRNA.