Difference between revisions of "Part:BBa K2442408"

Latest revision as of 03:26, 2 November 2017

Full split-GFP AND-gate circuit [Split GFP module 1 + Split GFP module 2]

This subproject is discussed in full detail on our [http://2017.igem.org/Team:Glasgow/ANDGate AND-Gate] wiki page.

A schematic representation of a final split-GFP AND-gate

This is the fully assembled split-GFP AND-gate. It is a composite part of the split GFP N-terminal "module 1" K2442405 and the C-terminal "module 2" K2442406

Both of these modules possess biobrick prefix errors prior to their protein coding sequences:

Sequencing of the PCR for the GFP C-terminal part revealed an error – the PCR design had incorporated an incorrect version of the BioBrick prefix.

Figure 2: A Biobrick prefix error in GFP C-term part.</b> Alignment of a sequencing read of the split GFP C-terminal to a reference sequence plasmid map. The bases highlighted in red indicate an insertion has occurred at those positions relative to the reference sequence. The insertion corresponds to the use of the “long” Biobrick prefix, rather than the short version which should precede ATG-starting parts. Alignments performed in ApE.

BioBrick parts beginning with ATG must use a shortened version of the prefix ending TAG, instead of the longer version for non-ATG parts which ends TAGAG. The short version ensures ideal spacing between upstream ribosome binding site parts and the start codon. Unfortunately, this oversight of the primer design was not initially noticed our plasmid map sequence for the C-terminal GFP part also contained the error. A part such as this with the extra two nucleotides before the start codon has the potential to be translated at decreased level from an upstream ribosome binding site.

The N-term GFP part was found to contain an extra A nucleotide immediately preceding the start-codon ATG. This erroneous nucleotide is present in the parts-registry sequencing for K1789003, but is not reported as an error. Unfortunately, now both split-GFP modules might be affected by improperly spaced ribosome binding sites. Despite this we proceeded to the next assembly stage.

A Biobrick prefix error in GFP N-term part.</b> Alignment of a sequencing read of the split GFP N-terminal to a reference sequence plasmid map. The base highlighted in red indicate an insertion has occurred at those positions relative to the reference sequence. The single-A insertion may correspond to the use a taq-polymerase during the original amplification of this coding sequence. Alignments performed in ApE.

Usage and Biology

For testing the final split-GFP AND-gate construct K2442408 was moved from E. coli DH5α to the strain DH5α-Z1. As described in the Methods section strains table, DH5α-Z1 contains an overexpressed version of the lacI gene, along with constitutively expressing tetR. These two genes allow for strict control of the lacI and tetR regulated promoters, which should be tightly repressed until the addition of their respective inducer molecules; IPTG and aTC (Anhydrotetracycline). Overnight cultures were prepared of the N-term, C-term, and Full Split-GFP AND-gate constructs; each in both DH5α and DH5α-Z1 strains. The following day these cultures were diluted 1 in 100 with fresh L-Broth, prepared along with a range of inducer and co-factor final concentrations. These conditions are detailed in Figure 7. 200 ul of these culture and inducer combinations were placed into wells of a clear-based black-sided 96 well plate. The prepared plate was then incubated shaking at 300 rpm within a BMG FLUOstar Omega plate reader, where cell growth (absorbance at 600 nm) and fluorescence (excitation at 485 nm and emission at 530 nm) readings taken at 1-hour intervals for 8 hours. 1 mM MgCl2 was added to the dual-inducer inductions as a cofactor which has been shown to improve the reliability of tetR-regulated promoter responses. As shown in Figure 7, no significant change in fluorescence between time 0 and 8 hours was detected in the full AND-gate construct, nor the individual modules. The fluorescence emission reading at 530 nm for the hour 0 timepoint was subtracted from the subsequent reading at the 8-hour timepoint. No noticeable difference in fluorescence can be observed between DH5α or the Z1 variant. A fluorescent response would have been expected in any of the 8 conditions containing both IPTG and aTC, in either strain, but this was not observed.

Figure 7: No fluorescence is observed from the split-GFP AND-gate during a wide array of induction conditions. Strongest induction would be expected in the 0.1mM IPTG, 100 ng/ml aTC condition.

The lack of fluorescent output from the split-GFP AND-gate could be explained by the errors found during DNA sequencing of the constructs. Both the N and C-terminal modules possessed improper spacing between the ribosome binding site and their ATG start codon. With more time it may have been possible to resurrect both final AND-gate constructs by using PCR mutagenesis to correct the sequence errors in each respective module.

Sequence and Features