Difference between revisions of "Part:BBa K3352004"

Latest revision as of 03:07, 26 October 2020

Strong Promoter and RBS SplintR Ligase Expressing Construct

The composite part utilizes a strong promoter (BBa_J23100), a ribosome binding site, SplintR ligase(BBa_K3352000), and a double terminator (BBa_K0015).

Figure 1: SplintR ligase with strong promoter, RBS, and double terminator

Construct Design

We optimized the DNA sequence for expression in E. coli and removed the PstI cutting site. We attached a 6x histidine tag (6x His-Tag) upstream of the SplintR ligase sequence for purification purposes followed by a glycine-serine linker (GS linker) to form our open reading frame (ORF) (BBa_K3352000). We flanked the open reading frame with an upstream strong promoter and strong ribosome binding site (RBS) combination (BBa_K880005) and downstream double terminator (BBa_B0015). This entire composite part was gene synthesized by IDT.

Results

Figure 2: Characterization of our Φ29 polymerase, parts BBa_K3352004 and BBa_K3352005, and SplintR ligase, BBa_K3352006 and BBa_K3352007.. All four constructs were ordered from Twist or IDT, conformed to a BioBrick assembly standard 10, and digested with EcoRI and PstI. Parts BBa_K3352004 and BBa_K3352005 were ordered from IDT and had a kanamycin backbone (pUCIDT KAN), which had a size of 2.7kB. BBa_K3352007 was also ordered from IDT, however, it contained an ampicillin backbone (pUCIDT AMP), which was also around 2.7kB. BBa_K3352006 was obtained from Twist Bioscience and was cloned into the ampicillin backbone (pSB1A3).

Characterization

Protein Expression and Purification

We transformed our designed plasmids (BBa_K3352004) into DH5⍺ E. coli cells. We grew overnight cultures, diluted those cultures, and then grew the cells to log phase. We lysed cells with xTractor Lysis Buffer (Takara Bio) and purified our His-tagged proteins using Ni sepharose affinity chromatography [2]. In order to check if our proteins were correct, we used SDS-PAGE.

Based on our results, our SplintR ligase construct that used a strong promoter and strong RBS combination (BBa_K3352004 and BBa_K3352005) did not express an appreciable amount of protein (Figure 3).

Figure 3: SDS-PAGE results show protein content at different steps of protein purification. A band around 35 kDa in the cell lysate (blue) and the eluate (red), matches our expected HIS-tagged Φ29. However, many other proteins were present in the eluate, and in the flowthrough lane (yellow). This prompted us to redesign our constructs.

References

1. Biolabs, N. E. (n.d.-c). SplintR® Ligase | NEB. Retrieved October 20, 2020, from https://international.neb.com/products/m0375-splintr-ligase

2. XTractorTM Buffer & xTractor Buffer Kit User Manual. (n.d.). 10.

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
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 851
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K3352004 short</partinfo>
-<h3> Construct Design </h3>
+The composite part utilizes a strong promoter (BBa_J23100), a ribosome binding site, SplintR ligase(BBa_K3352000), and a double terminator (BBa_K0015).
-This construct was created to express Splint R Ligase. Sequences used for the promoter, RBS, and terminator came from parts included in the iGEM Registry. This construct consists of a strong promoter and a strong RBS combination (BBKa_K880005), SplintR Ligase, and a downstream double terminator (BBa_BB0015).
+https://2020.igem.org/wiki/images/thumb/2/2b/T--TAS_Taipei--Parts_BBa_K3352004.png/800px-T--TAS_Taipei--Parts_BBa_K3352004.png
-<h3> Characterization </h3>
+<b>Figure 1: SplintR ligase with strong promoter, RBS, and double terminator</b>
-We used SDS-PAGE to check for SplintR Ligase expression in E. coli. We prepared overnight DH5&#9082; E. coli cultures and obtained OD600 readings to dilute cultures to standardized populations. We grew the cultures to log phase, and lysed cells with xTractor Lysis Buffer (Takara Bio). We purified our His-tagged proteins using Ni sepharose affinity chromatography, then ran our prepared samples through a nickel column in order to purify out our his-tagged proteins. However, the SDS-PAGE results showed that our SplintR Ligase did not express as strongly as we expected, which prompted us to redesign our constructs.
+<b><font size="+1.2"> Construct Design </font></b>
+We optimized the DNA sequence for expression in <i>E. coli</i> and removed the PstI cutting site. We attached a 6x histidine tag (6x His-Tag) upstream of the SplintR ligase sequence for purification purposes followed by a glycine-serine linker (GS linker) to form our open reading frame (ORF) (BBa_K3352000). We flanked the open reading frame with an upstream strong promoter and strong ribosome binding site (RBS) combination (BBa_K880005) and downstream double terminator (BBa_B0015). This entire composite part was gene synthesized by IDT.
+<b><font size="+1.2"> Results </font></b>
+https://2020.igem.org/wiki/images/4/4e/T--TAS_Taipei--Registry_1.png
+<b> Figure 2: Characterization of our Φ29 polymerase, parts BBa_K3352004 and BBa_K3352005, and SplintR ligase, BBa_K3352006 and BBa_K3352007.. All four constructs were ordered from Twist or IDT, conformed to a BioBrick assembly standard 10, and digested with EcoRI and PstI. Parts BBa_K3352004 and BBa_K3352005 were ordered from IDT and had a kanamycin backbone (pUCIDT KAN), which had a size of 2.7kB. BBa_K3352007 was also ordered from IDT, however, it contained an ampicillin backbone (pUCIDT AMP), which was also around 2.7kB. BBa_K3352006 was obtained from Twist Bioscience and was cloned into the ampicillin backbone (pSB1A3). </b>
+<b><font size="+1.2"> Characterization </font></b>
+<b><font size="+0.5"> Protein Expression and Purification </font></b>
+We transformed our designed plasmids (BBa_K3352004) into DH5⍺ <i>E. coli</i> cells. We grew overnight cultures, diluted those cultures, and then grew the cells to log phase. We lysed cells with xTractor Lysis Buffer (Takara Bio) and purified our His-tagged proteins using Ni sepharose affinity chromatography [2]. In order to check if our proteins were correct, we used SDS-PAGE.
+Based on our results, our SplintR ligase construct that used a strong promoter and strong RBS combination (BBa_K3352004 and BBa_K3352005) did not express an appreciable amount of protein (Figure 3).
+https://2020.igem.org/wiki/images/c/c5/T--TAS_Taipei--Registry_11.png
+<b> Figure 3: SDS-PAGE results show protein content at different steps of protein purification. A band around 35 kDa in the cell lysate (blue) and the eluate (red), matches our expected HIS-tagged Φ29. However, many other proteins were present in the eluate, and in the flowthrough lane (yellow). This prompted us to redesign our constructs. </b>
+<b><font size="+1.2"> References </font></b>
+. Biolabs, N. E. (n.d.-c). SplintR® Ligase | NEB. Retrieved October 20, 2020, from https://international.neb.com/products/m0375-splintr-ligase
+. XTractorTM Buffer & xTractor Buffer Kit User Manual. (n.d.). 10.