Team:CIDEB-UANL Mexico/labwork discussions

Here is the interpretation of the results that were obtained on all the experiments

Capture module - [Go to Results]

Ligation of NhaS and pSB1C3

The ligation transformed in E. coli of the NhaS module produced red and white colonies when we expected only red colonies (bacteria expressing the RFP). We did not know the reason of the unexpected result so we designed an experiment with the UV light promoter.

The NhaS module was proved in the experiment with the Petri Dishes in the UV camera. The red bacteria was already red (meaning that the RFP expression already started) before being exposed to the UV camera at 302nm. The promoter pUV 1765001 is activated by the UV exposition under 360 nm as the iGEM team Colombia_Israel 2007 reported. (iGEM Colombia_Isreal, 2006) The UV promoter does not have a reported wavelength interval. The unexpected result obtained can mean that the promoter is so sensible to the UV light that the normal UV radiation is enough to activate it without the need of being exposed to the UV camera. In the normal light the UV rays are present in different ammount as it is presented in the Global Solar UV Index publication. (World Health Organization, 2002) In the part description is reported that the UV promoter gets active and the protein must be expressed after 10 minutes of exposure in the UV camera, but after 2 hours there was no change in the white colonies so the RFP was not expressed there.

After doing the experiment we obtained that the red colonies continued with the RFP expression and the white colonies did not changed in color. As we expected to activate the RFP expression in the white colonies, this means that the time of the exposure was not enough to activate the UV promoter or the promoter did not work in the conditions we thought it would work. To see if the promoter was already activated we did another experiment.

The experiment with NaCl in different concentrations in Petri Dishes showed that the bacteria grew in a medium with high NaCl concentration. The control group with non-modified bacteria did not grow because it did not contain the Nhas insert so it was not able to survive in a saline medium. The white bacteria did actually grow but they did not expressed the RFP, but the fact that they did grow means that they have the NaCl resistance and the insert is inside them.

As the white and red colonies are supposed to come from the same ligation and to contain the same genetic information we need to prove that the insert was inside them. In order to prove this we sent samples of DNA to be sequenced to the DNA Synthesis and Sequentiation Biotechnology Institute Unit (USSDNA in Spanish), from the UNAM.

The primer used was in the complementary reverse chain, so the sequences are in the 3’ to 5’ direction. We did an analysis of the sequences obtained by aligning them with the BLAST Software.

The RFP sequence used in the alignment was the following (in 5' to 3' direction):

Atggcttcctccgaagacgttatcaaagagttcatgcgtttcaaagttcgtatggaaggttccgttaa
cggtcacgagttcgaaatcgaaggtgaaggtgaaggtcgtccgtacgaaggtacccagaccgctaaac
tgaaagttaccaaaggtggtccgctgccgttcgcttgggacatcctgtccccgcagttccagtacggt
tccaaagcttacgttaaacacccggctgacatcccggactacctgaaactgtccttcccggaaggttt
caaatgggaacgtgttatgaacttcgaagacggtggtgttgttaccgttacccaggactcctccctgc
aagacggtgagttcatctacaaagttaaactgcgtggtaccaacttcccgtccgacggtccggttatg
cagaaaaaaaccatgggttgggaagcttccaccgaacgtatgtacccggaagacggtgctctgaaagg
tgaaatcaaaatgcgtctgaaactgaaagacggtggtcactacgacgctgaagttaaaaccacctaca
tggctaaaaaaccggttcagctgccgggtgcttacaaaaccgacatcaaactggacatcacctcccac
aacgaagactacaccatcgttgaacagtacgaacgtgctgaaggtcgtcactccaccggtgcttaata
acgctgatagtgctagtgtagatcgctaa

It was aligned with the sequences obtained from the samples Nhas white bacteria and Nhas red bacteria.

NhaS sequence from white colonies (in 3' to 5' direction of the complementary reverse)

TAAATAAAAAGTTTTTTCTAATGCGTTTCTTCTCCTACAACCGAAAACACCGGGTCAGTGAGCGAGGA
ACCTGCATAACGCGAAGCACGCTTTTCCGCAAGAAGAAAAAGGGCAGGGTGGTGACACCTTGCCCTTT
TTTGCCGGACTGCAGCGGCCGCTACTAGTATTAGCGATCTACACTAGCACTATCAGCGTTATTAAGCA
CCGGTGGAGTGACTACCTTCAGCACGTTCGTACTGTTCAACGATGGTGTAGTCTTCGTTGTGGGAGGT
GATGTCCAGTTTGATGTCGGTTTTGTAAGCACCCGGCAGCTGAACCGGTTTTTTAGCCATGTAGGTGG
TTTTAACTTCAGCGTCGTAGTGACCACCGTCTTTCAGTTTCAGACGCATTTTGATTTCACCTTTCAGA
GCACCGTCTTCCGGGTACATACGTTCGGTGGAAGCTTCCCAACCCATGGTTTTTTTCTGCATAACCGG 
ACCGTCGGACGGGAAGTTGGTACCACGCAGTTTAACTTTGTAGATGAACTCACCGTCTTGCAGGGAGG
AGTCCTGGGTAACGGTAACAACACCACCGTCTTCGAAGTTCATAACACGTTCCCATTTGAAACCTTCC
GGGAAGGACAGTTTCAGGTAGTCCGGGATGTCAGCCGGGTGTTTAACGTAAGCTTTGGAACCGTACTG
GAACTGCGGGGACAGGATGTCCCAAGCGAACGGCAGCGGACCACCTTTGGTAACTTTCAGTTTAGCGG
TCTGGGTACCTTCGTACGGACGACCTTCACCTTCACCTTCGATTTTCGAACTCGTGACCGTTAACGGA
ACCTTTCCATACATGACCATGTTCTCTCGTCTGATTAGCATCGTGAGCCTGATTCTGTCCTTCTACTT
CGCTTACAAATACCGTTATCGTGTGATTAACGCGGTGCTGGGCCGTCGCTGGCTGCGTAAAGTTATTA
TCGGTTTTGCCATGCAGATTCCGATGATTCGTGACCGTATGCTGGGTAGCGTTCTGCAAAGTAACCGT
CCGCAAAATGTGTAA

NhaS sequence from red colonies (in 3' to 5' direction of the complementary reverse)

AAAGTGTCCACCCCGTACGACCGAGCGGAGCGAGTCAGTGAGCGAGGAAGCCTGCATAACGCGAAGTA
ATCTTTTCGGCTTAAAGAAAAAGGGCAGGGTGGTGACACCTTGCCCTTTTTTGCCGGACTGCAGCGGC
CGCTACTAGTATATAAACGCAGAAAGGCCCACCCGAAGGTGAGCCAGTGTGACTCTAGTAGAGAGCGT
TCACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGATGC
CTGGCTCTAGTAGCGATCTACACTAGCACTATCAGCGTTATTAAGCACCGGTGGAGTGACGACCTTCA
GCACGTTCGTACTGTTCAACGATGGTGTAGTCTTCGTTGTGGGAGGTGATGTCCAGTTTGATGTCGGT
TTTGTAAGCACCCGGCAGCTGAACCGGTTTTTTAGCCATGTAGGTGGTTTTAACTTCAGCGTCGTAGT
GACCACCGTCTTTCAGTTTCAGACGCATTTTGATTTCACCTTTCAGAGCACCGTCTTCCGGGTACATA
CGTTCGGTGGAAGCTTCCCAACCCATGGTTTTTTTCTGCATAACCGGACCGTCGGACGGGAAGTTGGT
ACCACGCAGTTTAACTTTGTAGATGAACTCACCGTCTTGCAGGGAGGAGTCCTGGGTAACGGTAACAA
CACCACCGTCTTCGAAGTTCATAACACGTTCCCATTTGAAACCTTCCGGGAAGGACAGTTTCAGGTAG
TCCGGGATGTCAGCCGGGTGTTTTAACGTAAGCTTTGGAACCGTACTGGAACTGCGGGGAACAGGATG
TCCCAAGCGAACGGCAGCGGACCACCTTTGGTAACTTTCAGTTTAGCGGTCTCGGGTACCTTCGAACG
GACGACCTTCACCTTCACCCTTCAATTTTCAAACTCGTGACCGTAAACGGAACCTTTCCATACAACTT
TGAAAACGCATGAAACTCATTTGAATAACGTCTTCCGGAAGAAAGCCCAATCTAAGTATTTTCTCCCT
CTTTTCTCATATAAATGTGATGAATATTTGATCTATCCGCCCTCCAACAACTTTCCCACAACAATCAT
GTATCGAAATTCCTGTTATACGACACTATAAAGATGGTATAAAAAGCCCGTGGAGGGGGCGTGACCA

Report

The report obtained from the analysis with the NhaS in red colonies is the following:

Image 1. Report generated by the software BLAST with the alignment of the RFP original sequence and the NhaS in red colonies sequence.

The RFP original sequence has a length of 709 bp. The match started at position 3 and ended at position 708, this means that almost all the RFP is present in the sample sequenced as we expected because the colonies were red.

The report obtained from the analysis with the NhaS in white colonies is the following:

Image 2. Report generated by the software BLAST with the alignment of the RFP original sequence and the NhaS in white colonies sequence.

The match ends at the 709 position from the original RFP sequence, but it did not start from the position 1 or 3, it starts at position 50. This means that there are 49 nucleotides that did not match with the original RFP sequence. This can be a possible cause in the problem with the RFP expression in white colonies, a mutation in the region of the RFP.

We also made an analysis with the Ribosome Binding Site (RBS) sequence:

Image 3. Report generated by the software BLAST with the alignment of the RBS original sequence and the NhaS in white (which presented 1 match) and red (which presented 2 matches) colonies sequence.

In the red colonies there where two matches, which is the complete sequence but in two parts: from 1 to 7 and from 6 to 12 positions. In the white colonies there was only one match, this means that the RBS sequence was not found there. If the RBS previous to the RFP has a problem, the mRNA cannot bind in the ribosome, and is not able to be translated.

Image 4. The region of the Biobrick at which a possible mutation could had happened.

With these results we can infer that the BioBrick works find and expresses the NhaS (because both of them survive in a high NaCl concentrated medium) but it stops being translated in the RBS or in the RFP region causing the colonies to be white instead of red but being able to survive in a medium with high NaCl concentration.

The question is if the problem is caused by a mutation, When did it happen?

The ligation transformed contained a DNA obtained from a digestion done the May 16th in the 3rd week registered in the Notebook. This digestion was exposed to the UV light camera at 302nm for about 5 minutes. After the digestion it was ligated and purified. Later it was transformed in E. coli. The chance of occurring a mutation of this insert was 1) before the miniPrep, inside the cell or 2) due to the UV radiation after the transformation. The UV radiation at 312nm can cause a damage in the DNA sample and to reduce the succes in transormation in E. coli (Gründemann, 1996). There are also several types of mutagenesis due to the UV radiation inside the cell (Ikehata & Ono, 2011) that can have occurred before the transformation to some samples of the plasmid. The red bacteria had the original DNA and the white bacteria had the mutated DNA.

UV experimentation

In this experiment it was expected the white bacteria to express the RFP protein to be equal as the red colonies. But after the experiment was done there was not a change and we can conclude this is because the white transformed bacteria had a mutation on the RBS region for the RFP gene leading to grown colonies without the RFP (red color) and according to the Department of Biochemistry, University of Turku, Finland when the ribosome-binding site (RBS) sequence, AGGAAA, was altered to AAGAAA, PPase production decreased to 19% of the wild-type. Surprisingly, when the RBS sequence was mutated to the consensus RBS sequence, AGGAGG, the intracellular levels of both ppa mRNA and PPase decreased drastically(Department of Biochemistry, 1991) meaning that a mutation in the RBS region leads to a malfunction of the gene and a decrease of the production of it.. The implications of these results are discussed.. This explains why we obtained two different transformed bacteria: the white and the red ones.

About the red colonies it can be stated that the expression of the RFP protein is due to the sensitiveness of the UV promoter, it means that the promoter is so sensitive that even though the plates were inoculated with no light they showed the red color. The interpretation is that there is still UV radiation that does not belong to visible light spectrum according to the Global Solar UV Index publication (World Health Organization, 2002) so the promoter was activated with the UV radiation present in the lab.

When the red bacteria were grown in a petri dish some white colonies grown as well along with the red bacteria, it can be concluded that two things happened: 1) At the moment of lifting bacteria from the petri dish to the tube some white bacterium came in the micropipette and were streaked in the petri dish with the red bacteria. 2) Also there is certain predisposition to mutation of the RBS of the GFP so white bacteria does not express RFP because of the mutation that occurred in the purification.

Viability test of the NhaS gene containing bacteria in salt - [Go to Results]

The first time the experiment one was performed, bacteria transformed with the capture plasmid were inoculated in Petri dishes with different concentrations of salt. All the bacteria transformed with NhaS in pSB1C3 (Red and White) lived in the 15% saline medium. This means that these bacteria have the resistance and support a medium with high NaCl concentration. All the control bacteria exposed to any concentration of salt died. This result supports the fact that the transformed bacteria with Nhas are more able to survive in a high NaCl concentrated medium. It means that this group (not transformed bacteria) did not have resistance to a medium with a high NaCl concentration. All the control bacteria inoculated only in LB medium (without salt) lived, as we expected because they were inoculated in normal conditions.

As in the experiment 1 was confirmed that the bacteria survived in a medium with LB and salt, in the experiment 2 the bacteria was inoculated with salt and without LB medium. The bacteria did not survived and this can be explained by the fact that the medium contains nutrients and substances the bacteria needs to survive.

In the experiment #3 after analyzing the data in tables, the results were positive, but inconclusive. With "positive but inconclusive" it is meant to say by this, is that the colonies with NhaS only displayed a higher number of colonies in the medium, while the other groups showed a higher death toll among bacteria expressed in less colonies. With inconclusive it is meant to say some colonies were contaminated with other groups from the same experimented bacteria and in some of these contaminated groups, only the contaminant (NhaS) survived while the other bacteria died. Only the NhaS only and control bacteria displayed a correlation in the data showed as a result of the experiments, so this same experiment is planned to be repeated to obtain better, more viable results in order to finally test the effectiveness of the nhas protein production.

Aroma module -

Characterization

The 3 sequences obtained from the DNA Synthesis and Sequentiation Biotechnology Institute Unit (Go to sequences) were aligned with the MEGA software and the complete original sequence was found in the 3 samples. This indicates that the BioBrick was cloned in E. coli and that the part was actually there.

Image 5. Alignment of the 3 sequenced samples, with the original sequence of the Aroma gene, done with the MEGA software

Qualitative experiment

People had different opinions while smelling the different Petri dishes with the aroma transformed bacteria. All of them used different words but at the end most of them remitted to the same meaning. The MIT team documented that the odor was produced when it was added 2mM of salicylic acid, so it was expected that the Petri dishes did not smell so much, but the results indicated that there was a mayor odor at 10mM than at 2mM (experiment previously done).

In the samples of 20 mM, both of the bacteria that was grown under 32 ºC had a little smell, which in theory, should not had happened because the riboswitch should be a loop at that temperature. One posible explanation is that the riboswitch is very sensitive to heat, and it could be activated during the time in which the Petri dishes were outside the incubator to perform the experiment and with the heat of the hands of the people who smelled it.

Finally, all of the samples that had a concentration of 30 mM of salicylic acid smelled like rotten food. By this, it can be inferred that all the bacteria in the Petri Dishes could not withstand the condition that particular concentration of salicylic acid, therefore they died, because “growth in a subinhibitory concentration of salicylic acid resulted in a significant reduction in the number of bacterial cells and a reduction in the rate of the number of bacteria increasing during logarithmic growth” (Bandara MB, et. al. 2006)

Bibliography/References

● Bandara MB, et al. (2006, October). Salicylic acid reduces the product... [Invest Ophthalmol Vis Sci. 2006] - PubMed

●Department of Biochemistry, University of Turku, Finland. (1991). Characterization of the 5' flanking region of the Escherichia coli ppa gene encoding inorganic pyrophosphatase: mutations in the ribosome-binding site decrease the level of ppa mRNA. 06/20/2014, of NCBI from http://www.who.int/uv/publications/en/GlobalUVI.pdf

● iGEM Colombian Team 2007 (2007, October 26). Part:BBa I765001. Retrieved June 15, 2014, from http://parts.igem.org/Part:BBa_I765001

● iGEM Colombian Team 2006 (2006). A Microbial Biosensor Device for Iron Detection under UV irradiation. Retrieved June 15, 2014, from https://2007.igem.org/wiki/index.php/IGEM_2006_Project

● NCBI. Retrieved June 13, 2014, from http://www.ncbi.nlm.nih.gov/pubmed/17003439

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