Difference between revisions of "Part:BBa K4006009"

Latest revision as of 23:44, 21 October 2021

MT-IEE5-arsC

Background

This composite part includes a coding sequence for the protein metallothionein and the protein arsenate reductase, 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.

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.

Figure 1. 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-ArsC 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. All four wells indicate successful cloning.

Figure 2. 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.

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 606
Illegal BamHI site found at 40
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI site found at 224
Illegal SapI.rc site found at 31

@@ Line 13: / Line 13: @@
 [[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-ArsC 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.
+This was confirmed with a restriction digest of the plasmid using XbaI and BstxI. MT-ArsC 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. All four wells indicate successful cloning.
-[[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. Each of the picked colonies (ftNa A-D) indicate three distinct bands as compared to the pASapI's four bands. The top band on each of the constructs is undigested plasmid due to the ineffective SapI enzyme.]]
+[[Image:T--ASU--afANDma.jpg|center|thumb|300px|<b>Figure 2.</b> Gel electrophoresis of restriction digest with XbaI and BstXI of the recombinant colonies.]]
 ===Transformation into ''Chlamydomonas reinhardtii''===