Part:BBa_K5122003
RBS -> guaA -> RBS -> guaB -> RBS -> aeBlue
We designed an insertion to be integrated into the overexpression plasmid. The insertion consists of sequences for genes guaA, guaB and aeBlue, each separated by spacer sequences and individual ribosome binding sites (RBS) to ensure proper expression. It has been proven that guaA and guaB belong to the same operon, so we designed them together under a single promoter. The insertion was integrated into the plasmid by two restriction sites: SacI and HindIII. The gene cassette was inserted between the inducible T7 promoter and terminator on the plasmid backbone. Once inside the bacterial cells, the gene expression was activated by the addition of IPTG as an inducer. Hence, guaA and guaB would be overexpressed, which ideally led to the overproduction of guanine. The aeBlue reporter gene serves as a visual marker to confirm successful transformation and expression. This design enables significant overexpression of the guaA and guaB gene products, thereby enhancing the formation of guanine crystals.
Fig. 1. Guanine synthesis pathway (from Jewett et al., 2009 J Bacteriol).
Engineering Cycle
Design
Construct designed in the first cycle aimed to boost production of guanine (GMP), It contained the genes GuaA and GuaB under the T7 promoter. These genes code the two enzymes (GMP synthetase and IMP dehydrogenase) that are involved in guanine synthesis pathway in bacteria. We hypothesised that GuaA and GuaB overexpression would lead to higher guanine production rate and thus more substrate for crystal formation. Additionally, the gene AeBlue was added to the construct to verify successful transformation and induction of the plasmid. BL21 E. coli was chosen as the host organism for our experiments due to well-established cultivation and transformation protocols.
Build
The pET45 plasmid backbone was amplified in TOP10 E. coli. The GuaA, GuaB and AeBlue containing insert was amplified by means of PCR using Phusion polymerase with a proof-reading activity. The length of the backbone and the amplicon was verified using gel electrophoresis. Next, DNA fragments of proper size were cut out from the gel and purified using a commercial gel extraction kit. Plasmid and insert were digested with SacI and HindIII restriction enzymes and ligated using T4 ligase according to enzyme supplier protocols. After the ligation, restriction digest was performed to verify the success of the protocols. Thereafter, the BL21 E. Coli was transformed with the construct.
Test
Transformed BL21 cells were grown to OD 0.6 (at 37 °C) in a liquid culture and then induced with IPTG. The cultures were grown in the presence of IPTG, at RT for 24 h in a liquid culture and also of IPTG+ agars. Samples from the liquid cultures were drawn at 2, 6 and 18 h post- IPTG induction in order to check for guanine concentration changes using Guanine Detection Kit and relative protein expression levels using SDS page of the sonicated bacterial lysates. Agar cultures were left to grow for a long time, since the formation of guanine crystals takes weeks in bacteria.
Target Gene Expression Verification
The target genes expression was confirmed by SDS-PAGE, where induced and uninduced E.coli culture lysates were run on an SDS-PAGE and band patterns of uninduced and IPTG-induced cultures several hours post-induction were compared to each other. Thick bands of sizes of 58.7 KDa (GuaA) and 52 KDa (GuaB) were observed only in the induced cultures, confirming target enzyme expression. There was no band corresponding to aeBlue observed.
Fig. 2. SDS-PAGE of induced and uniduced E. coli lysates.
Guanine Concentration Measurement
For guanine concentration measurement we utilized commercial Guanine Assay Kit (Cell Biolabs). For guanine concentration determination, a cell expansion was realized, first by an uninduced culture and secondly IPTG-induced cultures were established. Then the cultures were centrifuged and resuspended in PBS, then diluted until the OD was in the readable range (0.3-1). In this range, the concentration can be calculated as well as the volume necessary to realize solutions of the concentration necessary for the assay kit (1-2 * 10⁶ cells/mL in PBS). The samples are sonicated and the assay was realized. The results obtained from the assay were not conclusive as the standard measurements were not adequate due to unknown reasons. The test was repeated 3 times with three different kits (two colometric and one fluorescence).
Learn
Despite good culture growth and seemingly successful cloning steps, we did not observe AeBlue+ / blue color of our liquid culture nor of the colonies. As for the SDS-PAGE results, thicker bands of for GuaA (58,665 Da) and GuaB (52,022 Da) proteins were observed in the IPTG-induced lysates (as compared to the uninduced control lysates) but no band corresponding to AeBlue chromoprotein (25 900 Da) was observed. Thus, we went back to the construct design to revise the genetic components placement, inter-gene regions design and orientations on the genes. We have also isolated the plasmid and sent it for sequencing in order to gain assurance in the construct integrity.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 252
Illegal AgeI site found at 1057
Illegal AgeI site found at 2013
Illegal AgeI site found at 2601 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 2301
//function/biosynthesis
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