Difference between revisions of "Part:BBa K4879006"

Latest revision as of 18:35, 11 October 2023

JcFATA expression construct

The transcriptional unit coding for JcFATA, designed for expression in Y. lipolytica.

The expression cassette was assembled along with the JcFATB and the CvFAP expression cassettes with the help of the NEBuilder HiFi DNA Assembly kit.

Characterization

Cloning: The assembled gene(s) were then inserted into the pYLEX plasmid (sourced from the Yarrowia Lipolytica Expression Kit, procured from Yeastern Biotech), by the means of NEBuilder HiFi DNA Assembly. The expected clones were then digested with NotI and SalI. The resulting digestion product is run on a 0.8% agarose gel. The cloning was confirmed, with the cloning success also being verified with sequencing. The linearized fragment was expected to be at above the 10 kbp band in the ladder (~13,000 bp)

Fig: Lanes 1-4 contain the possible clones and lane 5 contains the ladder. Clones in lanes 1,3, and 4 are likely successful clones.

Expression: To verify the expression of JcFATA, the coding sequence had a 6xHis tag attached to the C-terminus. Subsequently, a Western blot was done for the protein after normalization.

The desired bands were visible in the blot but in the control too, as a result of possible contamination in the control. Thus, further experiments are needed to troubleshoot the problem and characterize the part fully. Depending on the refined results of the experiments, the functional validity of the part will be confirmed for future use.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal prefix found in sequence at 1642
Illegal suffix found in sequence at 1912
Illegal EcoRI site found at 1906
Illegal PstI site found at 862
Illegal PstI site found at 1469
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 1642
Illegal EcoRI site found at 1906
Illegal SpeI site found at 1913
Illegal PstI site found at 862
Illegal PstI site found at 1469
Illegal PstI site found at 1927
Illegal NotI site found at 1648
Illegal NotI site found at 1920
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 1642
Illegal EcoRI site found at 1906
Illegal BglII site found at 1311
23
INCOMPATIBLE WITH RFC[23]
Illegal prefix found in sequence at 1642
Illegal suffix found in sequence at 1913
Illegal EcoRI site found at 1906
Illegal PstI site found at 862
Illegal PstI site found at 1469
25
INCOMPATIBLE WITH RFC[25]
Illegal prefix found in sequence at 1642
Illegal EcoRI site found at 1906
Illegal XbaI site found at 1657
Illegal SpeI site found at 1913
Illegal PstI site found at 862
Illegal PstI site found at 1469
Illegal PstI site found at 1927
1000
COMPATIBLE WITH RFC[1000]

@@ Line 5: / Line 5: @@
 The transcriptional unit coding for JcFATA, designed for expression in <i>Y. lipolytica</i>.
-The expression cassette was assembled together along with the JcFATB and the CvFAP expression cassettes with the help of the NEBuilder HiFi DNA Assembly kit.
+The expression cassette was assembled along with the JcFATB and the CvFAP expression cassettes with the help of the NEBuilder HiFi DNA Assembly kit.
 ===Characterization===
@@ Line 15: / Line 15: @@
 Fig: Lanes 1-4 contain the possible clones and lane 5 contains the ladder. Clones in lanes 1,3, and 4 are likely successful clones.
+<b>Expression:</b>
+To verify the expression of JcFATA, the coding sequence had a 6xHis tag attached to the C-terminus. Subsequently, a Western blot was done for the protein after normalization.
+<html><img src="https://static.igem.wiki/teams/4879/wiki/his-blot.jpg"width="720"height="720"></html>
+The desired bands were visible in the blot but in the control too, as a result of possible contamination in the control. Thus, further experiments are needed to troubleshoot the problem and characterize the part fully. Depending on the refined results of the experiments, the functional validity of the part will be confirmed for future use.