Difference between revisions of "Part:BBa K1639014"

Latest revision as of 09:56, 22 September 2015

TnrA-pAlsT-mRFP reporter system

Main goal of this part is to show TnrA protein’s repressor activity on pAlst promoter and derepression of this promoter in the presence of NH3. Thereby we combined TnrA protein’s and pAlst’s gene sequences on the same biobrick. As a reporter, we put RFP gene sequence in front of the pAlst sequence. To make sure the repressive protein TnrA is produced in a high amount, we combined TnrA-pAlst-RFP gene sequence with T7 promoter which has a high transcription rate.Also, we put a Lac operator which LacI protein can bind, between this sequence and T7 promoter, in order to create a controllable system. We searched to find an expression vector working in harmony with this system, peT45-b expression vector was serving this purpose. . When we ordered our genes, we put RFC10 prefix site on 3’ end of TnrA-pAlst-RFP gene sequence and also BamHI restriction enzyme recognition site for cloning to peT45-b. We added RFC10 suffix site on 5’ end of the same gene sequence and also XhoI restriction enzyme recognition site for cloning to peT-45 vector, again.

Figure 1

As shown above,we planned to clone the ordered TnrA-pAlst-RFP gene into peT45-b vector by using BamHI and XhoI enzymes. Final construct after this cloning is, T7 promoter-Lac operator(LacO)-HisTag-TnrA-pAlst-RFP, respectively. Besides, a constitutive promoter called LacI promoter and LacI protein sequence in front of that, found on peT45-b vector’s another part. It is obvious that this part is IPTG-dependent, so Western Blot can be performed easily with the help of His Tag.

Figure 2

According to this system, under the absence of IPTG; TnrA production will be repressed, pAlsT promoter will be derepressed and RFP won’t be produced. If IPTG is present, TnrA will be produced , pAlsT promoter becomes derepressed and red colored bacteria will be observed with production of RFP. After showing stability of TnrA-pAlst sytem by using the RFP fluorescence protein, Toehold-TEV protease will be replaced with RFP for binding to AND gate system.

Figure 3

Usage and Biology

Figure 1: If there is a sufficient amount of NH3, Toehold and TEV protease will be produced. If there isn’t a sufficient amount of NH3, Toehold and TEV Protease will not be produced.

Bacteria use nitrogen which is present in nearly all macromolecules such as proteins, carbonhydrates and peptidoglycan. Prokaryotes have developed transport and assimilation systems for a variety of nitrogen sources for living under optimal conditions and regulate their own systems. This regulatory network allows an adequate response to situations of nitrogen limitation.

In the Bacillus subtilis, ammonium assimilation occurs via the glutamine synthetase - glutamate synthase pathway. Bacillus subtilis faces nitrogen- limiting conditions when it consumes glutamate as a prior nitrogen source, while glutamine is the secondly preferred nitrogen source.

Two transcription factors, TnrA and GlnR, and one enzyme, the Glutamine Synthase, are the major players in the B. Subtilis nitrogen regulatory network. We use TnrA transcription factor in our system.

Under nitrogen-limited conditions, TnrA works as an activator and a repressor both. TnrA represses expression of glnRA (Glutamine Synthase) [1], gltAB (Glutamate Synthase) [2] and other genes. Also the form of Glutamine Synthase which is feedback inhibited by excess glutamine, directly interacts with and unbinds from TnrA, thus blocks its DNA-binding activity [3]. Based on all this information, if the amount of NH3 is not sufficient, glutamine synthase will not work properly, glutamine will be produced in a low amount, TnrA will bind to promoter and Toehold production will be repressed. But if there is a sufficient amount of NH3, glutamine will be produced in a high level, TnrA will not repress the promoter as previous and Toehold – Tev Protease will be produced in a high amount.

Seventeen TnrA targets were detected by a combination of DNA microarray hybridization, a genome-wide search for TnrA boxes, and gel retardation assays [4]. The TnrA box consensus delimited in this study to a 17- bp interrupted, inverted repeat sequence, TGTNANAWWWTNTNACA.

Characterization

pSB1C3-TnrA-pAlst-RFP CLONING

We cloned IDT G-Blocks GadE gene into PSB1C3 vector in order to make it ready to be submitted and have many copies of it. For this purpose we digested PSB1C3 vector and TnrA-pAlst-RFP G-Blocks with ECoRI and PstI restriction enzymes. Then we ligated these cut genes into the plasmid by using T4 DNA Ligase . Ocurring products were transformed into BL21 competent cell strain.

To check if the cloning is correct, a colony PCR was perfomed with Verify Forward and Verify Reverse primers. If the cloning isn’t made properly t the band should be 314 bp long, but if colony PCR worked, then bands should be 1800 bp long.

Figure 4

As the result of Colony PCR experiments, we observed bands are either negative or less molecule-weighted than we expected. We made liquid cultures of the not negative colonies, and isolated DNA from these colonies after incubating for 16 hours. we did cut-check with EcoRI and PstI enzymes in order to control occurring DNA’s.

After cut-check, we observed less molecule-weighted bands again. Therefore we decided to control our ordered G-Blocks and did a PCR experiment with CMV forward and SV40 reverse primers. The gel image of this PCR is shown above.

TnrA-pTnrA-RFP PCR

Figure 5

After that PCR, we observed less molecule-weighted bands than normal.

pET45-TnrA-pTnrA-RFP CLONNING

We questioned if the plasmid is incorrect because the results of PSB1C3 cloning weren’t matching with our expected results. We cut the G-Block with BamHI and XhoI and ligated it with pET45-b which was cut with same enzymes. We transformed this ligated product into BL21 bacteria strain, which has T7 RNA polymerase.

In order to control if the cloning is made correctly, we did a colony PCR with T7 Promoter reverse and T7 promoter forward. If cloning isn’t made properly, the band should be at 360 bp line and if is made properly the band should be at 1779 bp line.

In colony PCR results, we observed lower molecule-weighted bands than expected, again. In this case we realized that this gene sequence was wrong.

We didn’t have enough time to order and clone this gene so we didn’t continue to do this part’s experiments.

Figure 6

Sequence and Features

References

[1]: Brown SW, Sonenshein AL. Autogenous regulation of the Bacillus subtilis glnRA operon. J Bacteriol. 1996;178(8):2450-4
[2]: Commichau FM, Wacker I, Schleider J, et al. Characterization of Bacillus subtilis mutants with carbon source-independent glutamate biosynthesis. J Mol Microbiol Biotechnol. 2007;12(1-2):106-13.
[3]: Wray LV, Zalieckas JM, Fisher SH. Bacillus subtilis glutamine synthetase controls gene expression through a protein-protein interaction with transcription factor TnrA. Cell. 2001;107(4):427-35.
[4]: Yoshida K, Yamaguchi H, Kinehara M, Ohki YH, Nakaura Y, Fujita Y. Identification of additional TnrA-regulated genes of Bacillus subtilis associated with a TnrA box. Mol Microbiol. 2003;49(1):157-65.