Difference between revisions of "Part:BBa K2842669"

Revision as of 17:37, 17 October 2018

mScarlet reporter with TerL-C intein on the N terminus

This DNA construct encodes a C terminal segment of the AceL-TerL intein fused to the N terminus of a mScarlet reporter protein which contains a C terminal StrepTag for purification. The AceL-TerL intein acts as a protein ligase that forms a peptide bond between mScarlet and another protein bound to a complementary split intein. During this process the intein splices itself out of protein and is not present in the final fusion protein. This construct is a modular platform for the creation of split intein fusion proteins through the SapI restriction sites located immediately upstream and downstream of the mScarlet reporter.BBa_K2832680 was designed to be used in conjunction with this construct as it contains the corresponding N terminal intein segment to enable intein trans-splicing.

Figure 1: Comparisons of fluorescence/OD600 of BBa_K2842669 and BBa_K1362101

(1-Red) BBa_K2842669 in BL21*(DE3) cells induced with 400 μM IPTG
(2-Orange) BBa_K2842669 in BL21*(DE3) cells without IPTG induction
(3-Grey) BBa_K1362101 expressed in BL21*(DE3) cells />

Experimental Results

This construct (BBa_K2842669) is also an improvement of the part BBa_K1362101. Both parts were designed to allow for the modular design and assembly of intein fusion proteins, but our part has distinct advantages and improvements. We used mScarlet as our reporter instead of mRFP1 as it is known to be brighter than mRFP1 and a fast folder. Our reporter protein mScarlet has an 11-fold increase in fluorescence/OD600 compared to BBa_K1362101 when driven by the expression from the T7 promoter (Fig3). Our construct also has a dual function as both an assembly construct and reporter for building new inteins but as an intein fusion by itself. Due to this, it can be used for both to test intein functionality in other constructs and to build new ones.

We measured the expression of BBa_K2842669 with a BMG Fluostar plate reader by analysis of it's fluorescence at varying IPTG concentrations. We found that mScarlet is very well expressed when produced from an IPTG-inducible T7 promoter. Increasing IPTG concentrations from 400 uM to 800 uM had little effect on expression levels and cell growth rate (Fig1A, Fig1B), indicating that IPTG concentrations at 400 uM are sufficient to overproduce our protein. We also tested the modularity of the construct by replacing mScarlet with with two new sequences to create the biobricks BBa_K2842710 and BBa_K2842720. We have confirmed this assembly with Sanger sequencing.

Figure 2: Expression of IP at varying IPTG concentrations Initial experiments suggested that 400 uM of IPTG was optimal for expression(data not shown). A plate reader was used to examine the region of IPTG concentrations around 400 uM </p>

Purification of BBa_K2832669

SDS-PAGE of BBa_K2832669

(L) Protein standard numbers are in kDa (1) The clear lysate of BBa_K2832669 before purification (2) After purification on Streptactin columns

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Sequence and Features