Difference between revisions of "Part:BBa K4006001"

Revision as of 20:53, 21 October 2021

arsC

Background

This part contains a protein coding sequence for arsenate reductase that has been codon optimized for use in the chloroplast of Chlamydomonas reinhardtii. Arsenate reductase catalyzes the reduction of arsenate [As(V)] to arsenite [As(III)]. Using 2 µM arsenate at a substrate, the Kcat of the protein has been recorded as 4.5 min(-1). It was introduced into our plasmid backbone MT-pASapI via golden gate assembly.

Cloning into E. coli and Verification

They were transformed into 5-alpha 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 left is the experimental transformation of arsC into plasmid backbone MT-pASapI using Golden Gate Assembly. Multiple individual colonies are present on the plate, indicating successful transformation. The plate on the right is the negative control. Little to no colonies are present on the plate, indicating that there should not be high background or incomplete recombination in our experimental plate.

This was confirmed with a restriction digest of the plasmid using XbaI and BstxI. ArsC was expected to have two bands of sizes 4.4kb and 2.6kb. This is a contrast to the MT-pASapI backbone, which would result in two bands of 4.4kb and 2.3kb. The gel showed three bands for all of the constructs run, the top band being undigested plasmid due to ineffective restriction digest. However, the other bands showed that the inserts contained bands of the appropriate size, and were different from the MT plasmid.

Figure 2. Gel electrophoresis of restriction digest with XbaI and BstXI of original pASapI plasmid, the backbone plasmid with integrated MT used for assembly, and the recombinant colonies with arsC in place of MT. Each of the picked colonies (arsC 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.

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
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_K4006001 short</partinfo>
-This part contained a protein coding sequence for arsenate reductase that has been codon optimized for use in the chloroplast of Chlamydomonas reinhardtii. Arsenate reductase catalyzes the reduction of arsenate [As(V)] to arsenite [As(III)]. Using 2 µM arsenate at a substrate, the Kcat of the protein has been recorded as 4.5 min(-1).
+===Background===
-We were able to clone this construct into our plasmid and select for transformed E. coli colonies.
+This part contains a protein coding sequence for arsenate reductase that has been codon optimized for use in the chloroplast of ''Chlamydomonas reinhardtii''. Arsenate reductase catalyzes the reduction of arsenate [As(V)] to arsenite [As(III)]. Using 2 µM arsenate at a substrate, the Kcat of the protein has been recorded as 4.5 min(-1). It was introduced into our plasmid backbone MT-pASapI via golden gate assembly.
-https://2021.igem.org/wiki/images/3/36/T--ASU--arsCplates.jpg
+===Cloning into ''E. coli'' and Verification===
-Digestion of the miniprepped DNA in the plasmid pASapI with XbaI and BstXI should result in two bands of approximate sizes 4446 bp and 2658 bp as compared to the original plasmid which should have three bands of sizes 4446, 1376, and 800 bp. The top band in each of the columns is undigested DNA, indicating successful cloning of each of the four colonies.
+They were transformed into 5-alpha competent ''E. coli'' cells from NEB. The cloning was successful, as the positive control plate showed significantly more colonies than the negative control plate.
-https://2021.igem.org/wiki/images/9/93/T--ASU--arsCGel.jpg
+[[Image:T--ASU--arsCplates.jpg|center|thumb|700px|<b>Figure 1.</b> The plate on the left is the experimental transformation of arsC into plasmid backbone MT-pASapI using Golden Gate Assembly. Multiple individual colonies are present on the plate, indicating successful transformation. The plate on the right is the negative control. Little to no colonies are present on the plate, indicating that there should not be high background or incomplete recombination in our experimental plate.]]
-Unfortunately, we were unable to successfully transform this construct into C. reinhardtii and test expression or effect on arsenic uptake in the algae.
+This was confirmed with a restriction digest of the plasmid using XbaI and BstxI.  ArsC was expected to have two bands of sizes 4.4kb and 2.6kb. This is a contrast to the MT-pASapI backbone, which would result in two bands of 4.4kb and 2.3kb. The gel showed three bands for all of the constructs run, the top band being undigested plasmid due to ineffective restriction digest. However, the other bands showed that the inserts contained bands of the appropriate size, and were different from the MT plasmid.
+[[Image:T--ASU--arsCgel.jpg|center|thumb|300px|<b>Figure 2.</b> Gel electrophoresis of restriction digest with XbaI and BstXI of original pASapI plasmid, the backbone plasmid with integrated MT used for assembly, and the recombinant colonies with arsC in place of MT. Each of the picked colonies (arsC 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.]]
+===Transformation into ''Chlamydomonas reinhardtii''===
+We were unable to successfully transform this construct into ''C. reinhardtii''.
 <!-- Add more about the biology of this part here