Difference between revisions of "Part:BBa K4006013"

Latest revision as of 00:32, 22 October 2021

MT-6XHis-IEE5-arsC-HA-IEE2-FLAG-fTNa

Background

This composite part includes coding sequences for the proteins metallothionein, arsenate reductase, and the protein ferritin, all codon optimized for use in the Chlamydomonas reinhardtii chloroplast. They were also each tagged with a different epitope tag, for the eventual purpose of protein expression evaluation by Western blot. The intention was for them to work synergistically to improve the uptake of arsenic out of contaminated water.

Cloning into E. coli and Verification

The constructs were designed by our team and produced by IDT. They were transformed into 10-Beta competent E. coli cells from NEB. The “Tagged” sequence, MT/6xHIS-IEE2-ArsC/HA-IEE5-FtnA/FLAG, was designed by our team and produced in two parts by IDT. However, our team did not have any success cloning this construct via golden braid. This may be due to the presence of IEE2, which is also present in the pASapI backbone. We were unable to confirm cloning via restriction digest and often did not get significant results from the transformation into 10-beta competent E. coli cells. Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 589
Illegal BamHI site found at 45
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI site found at 5
Illegal SapI site found at 685
Illegal SapI site found at 1750
Illegal SapI.rc site found at 233
Illegal SapI.rc site found at 1150
Illegal SapI.rc site found at 2305

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 __NOTOC__
 <partinfo>BBa_K4006013 short</partinfo>
 ===Background===
-This composite part includes a coding sequence for the protein metallothionein and the protein ferritin, both codon optimized for use in the ''Chlamydomonas reinhardtii'' chloroplast. The intention was for them to work synergistically to improve the uptake of arsenic out of contaminated water.
+This composite part includes coding sequences for the proteins metallothionein, arsenate reductase, and the protein ferritin, all codon optimized for use in the ''Chlamydomonas reinhardtii'' chloroplast. They were also each tagged with a different epitope tag, for the eventual purpose of protein expression evaluation by Western blot. The intention was for them to work synergistically to improve the uptake of arsenic out of contaminated water.
 ===Cloning into ''E. coli'' and Verification===
-The constructs were designed by our team and produced by IDT. They were transformed into 10-Beta competent ''E. coli'' cells from NEB. The cloning was successful, as the positive control plate showed significantly more colonies than the negative control plate.
+The constructs were designed by our team and produced by IDT. They were transformed into 10-Beta competent ''E. coli'' cells from NEB. The “Tagged” sequence, MT/6xHIS-IEE2-ArsC/HA-IEE5-FtnA/FLAG, was designed by our team and produced in two parts by IDT. However, our team did not have any success cloning this construct via golden braid. This may be due to the presence of IEE2, which is also present in the pASapI backbone. We were unable to confirm cloning via restriction digest and often did not get significant results from the transformation into 10-beta competent ''E. coli'' cells.
-[[Image:T--ASU--mtarscplate.png|center|thumb|700px|<b>Figure 1.</b> The plate on the right is the experimental transformation of the part into plasmid backbone MT-pASapI using Golden Gate Assembly. Multiple individual colonies are present on the plate, indicating successful transformation. The plate on the left is the negative control. The colonies present may indicate a high background or low efficacy of our initial digest with SapI.]]
-This was confirmed with a restriction digest of the plasmid using XbaI and BstxI. MT-FTNA was expected to have two bands of 4.4kb and 3.7kb. The number and size of bands shown in the gel were as expected, and indicated that the plasmid had been successfully integrated with our combined parts. This is a contrast to the MT-pASapI backbone, which should result in two bands of 4.4kb and 2.3kb.
-[[Image:T--ASU--ftnaGel.png|center|thumb|300px|<b>Figure 2.</b> Gel electrophoresis of restriction digest with XbaI and BstXI of the recombinant colonies.]]
-===Transformation into ''Chlamydomonas reinhardtii''===
-We were unable to successfully transform this construct into ''C. reinhardtii''.
 <span class='h3bb'>Sequence and Features</span>
 <partinfo>BBa_K4006013 SequenceAndFeatures</partinfo>