Difference between revisions of "Part:BBa K4472990"

Latest revision as of 03:16, 12 October 2022

Split ribozyme detecting kanamycin and expressing GFP

This composite part is build out of the split ribozyme halfs, guide RNAs complementary to kanamycin resistance mRNA and a GFP reporter. We assembled this part using golden gate assembly and transformed it into E.coli DH5alpha.

In our colony PCRs we were able to confirm that we have assembled the construct correctly as all our PCRs had bands at the expected height of around 1600-1700 bp.

Figure 1: Gel of Colony PCR of assembled split ribozyme - reporter constructs. Bands expected at around 1600-1700bp. Ladder: 1kb benchtop from Promega. Marked is the PCR of 2 clones with BBa_K4472990.

The promoters were too weak to get any signal in our plate reader measurements afterwards. Our model showed that stronger promoters would have had a much stronger effect, see here: https://2022.igem.wiki/uni-hamburg/model

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 331
Illegal NheI site found at 354
Illegal NheI site found at 768
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 670
Illegal XhoI site found at 53
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 1495

@@ Line 10: / Line 10: @@
 https://static.igem.wiki/teams/4472/wiki/parts/split-ribozym-colony-pcr-kan.png
 </div>
-Figure 1: Gel of Colony PCR of assembled split ribozyme - reporter constructs. Bands expected at around 1600-1700bp. Lassder: 1kb benchtop from Promega. Marked is the PCR of 2 clones with BBa_K4472990.
+Figure 1: Gel of Colony PCR of assembled split ribozyme - reporter constructs. Bands expected at around 1600-1700bp. Ladder: 1kb benchtop from Promega. Marked is the PCR of 2 clones with BBa_K4472990.