Team:CIDEB-UANL Mexico/labwork results

Biobricks construction- Return to the top

Here are the results of how the team got the modules of the project

Minipreps

Digestions

Purification

Ligations

Characterization

Minipreps- Return to the top

The first step to build up the four modules in bio-brick format with the vector pSB1C3 is to isolate the plasmid DNA from the bacteria through a mini prep.

The next electrophoresis geles (Image 1) shows that the extraction of the DNA was performed correctly. As in the beginning it was planned to put the four modules in vectors with different antibiotic resistance (such as pSB1C3, pSB1T3, pSB1A3 and pSB1K3) in a single E. col, but the team decided to first insert all of the genes in pSB1C3 so they could be sent to the parts registry.

Digestions-Return to the top

The next step of obtaining the DNA is get only the needed fragment for the project, this would be accomplished through a digestion with the enzymes of the biobrick format: EcoRI and PstI (In the case of pSB1C3, nhaS and BSMT1 opt.). But in the case of the union module, there is a fusion protein, it means that the digestion of AIDA and L2 (parts of the fusion protein) is with other enzymes, the first with PstI and BglII, and the second with EcoRI and BamHI.

In the digestion made in order to get the plasmid pSB1C3 and the gene NhaS (Image 2. right gel) both were differentiated from their respective insert (RFP) and plasmid (pUC57) by its length. The plasmid stays in the upper part of the gel while the insert stays below it.

The same happened with the digestion of BSMT1 opt., AIDA and L2 (Image 2. left gel) the genes were differentiated by its length.

Purification- Return to the top

There is not need of make all the purification process of the fragments gotten in the digestion, easily the ligation can be done. But to make sure that only the pieces that were wanted to ligate, were together, it was done a purification. Then to confirm that there were only the fragments such as pSB1C3, BSTM1 opt. L2 and AIDA, it was made an electrophoresis gel:

Ligations- Return to the top

After the purification of the digestions was made, the fragments were ligated with their respective gene-plasmid. Then bacteria were transformed with those ligations and inoculated in a Petri dish obtaining the next result:

Ligation of NhaS and pSB1C3 [Capture Module] - Return to the top

In the first transformation of the ligation of NhaS and pSB1C3 grew red (expressing RFP) and white bacteria. The Petri dish was not cover by aluminum after its inoculation.

There was a second transformation of the same ligation, also it was inoculated in a Petri dish getting Image 5. as result. There were also present red and white bacteria. The Petri dish was not cover by aluminum after its inoculation.

Result of ligation BSMT1 (optimized) and pSB1C3 [Aroma Module] - Return to the top

The main gene of the aroma module, BSMT1 opt. was ligated with pSB1C3, and then transformed in bacteria in order to be inoculated. In the resulting inoculation there were only white colonies of bacteria.

Characterization- Return to the top

Capture module characterization

Once NhaS was in pSB1C3, it was needed to prove it through a characterization, also there was the question of why in the ligation there were red and white bacteria, if all had the plasmid to chloramphenicol resistance (reason they lived). Which of the two types really had NhaS?. To know with which enzyme make the digestion, it was made a digital digestion of the plasmid and the insert getting the next result:

The problem was that the enzyme that cuts NhaS and pSB1C3 was not available to the team, and it would take a long time to get it. To solve this problem, it was sent the DNA to be sequenced and then prove that the ligation actually occurred, and NhaS was inside pSB1C3. It was used a primer that is from 5' to 3' in the complementary chain:

5´ATTACCGCCTTTGAGTGAGC 3'

3' AAAGTGTCCACCCCGTACGACCGAGCGGAGCGAGTCAGTGAGCGAGGAAGCCTGCATAACGCGAAGTAATCTTTTCGGC
TTAAAGAAAAAGGGCAGGGTGGTGACACCTTGCCCTTTTTTGCCGGACTGCAGCGGCCGCTACTAGTATATAAACGCAGAAA
GGCCCACCCGAAGGTGAGCCAGTGTGACTCTAGTAGAGAGCGTTCACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTT
TCGACTGAGCCTTTCGTTTTATTTGATGCCTGGCTCTAGTAGCGATCTACACTAGCACTATCAGCGTTATTAAGCACCGGTG
GAGTGACGACCTTCAGCACGTTCGTACTGTTCAACGATGGTGTAGTCTTCGTTGTGGGAGGTGATGTCCAGTTTGATGTCGG
TTTTGTAAGCACCCGGCAGCTGAACCGGTTTTTTAGCCATGTAGGTGGTTTTAACTTCAGCGTCGTAGTGACCACCGTCTTT
CAGTTTCAGACGCATTTTGATTTCACCTTTCAGAGCACCGTCTTCCGGGTACATACGTTCGGTGGAAGCTTCCCAACCCATG
GTTTTTTTCTGCATAACCGGACCGTCGGACGGGAAGTTGGTACCACGCAGTTTAACTTTGTAGATGAACTCACCGTCTTGCA
GGGAGGAGTCCTGGGTAACGGTAACAACACCACCGTCTTCGAAGTTCATAACACGTTCCCATTTGAAACCTTCCGGGAAGGA
CAGTTTCAGGTAGTCCGGGATGTCAGCCGGGTGTTTTAACGTAAGCTTTGGAACCGTACTGGAACTGCGGGGAACAGGATGT
CCCAAGCGAACGGCAGCGGACCACCTTTGGTAACTTTCAGTTTAGCGGTCTCGGGTACCTTCGAACGGACGACCTTCACCTT
CACCCTTCAATTTTCAAACTCGTGACCGTAAACGGAACCTTTCCATACAACTTTGAAAACGCATGAAACTCATTTGAATAAC
GTCTTCCGGAAGAAAGCCCAATCTAAGTATTTTCTCCCTCTTTTCTCATATAAATGTGATGAATATTTGATCTATCCGCCCT
CCAACAACTTTCCCACAACAATCATGTATCGAAATTCCTGTTATACGACACTATAAAGATGGTATAAAAAGCCCGTGGAGGG
GGCGTGACCA 5'

And this is the sequence obtained from the miniPrep of the white bacteria transformed with NhaS:

3' TAAATAAAAAGTTTTTTCTAATGCGTTTCTTCTCCTACAACCGAAAACACCGGGTCAGTGAGCGAGGAACCTGCATAAC
GCGAAGCACGCTTTTCCGCAAGAAGAAAAAGGGCAGGGTGGTGACACCTTGCCCTTTTTTGCCGGACTGCAGCGGCCGCTAC
TAGTATTAGCGATCTACACTAGCACTATCAGCGTTATTAAGCACCGGTGGAGTGACTACCTTCAGCACGTTCGTACTGTTCA
ACGATGGTGTAGTCTTCGTTGTGGGAGGTGATGTCCAGTTTGATGTCGGTTTTGTAAGCACCCGGCAGCTGAACCGGTTTTT
TAGCCATGTAGGTGGTTTTAACTTCAGCGTCGTAGTGACCACCGTCTTTCAGTTTCAGACGCATTTTGATTTCACCTTTCAG
AGCACCGTCTTCCGGGTACATACGTTCGGTGGAAGCTTCCCAACCCATGGTTTTTTTCTGCATAACCGGACCGTCGGACGGG
AAGTTGGTACCACGCAGTTTAACTTTGTAGATGAACTCACCGTCTTGCAGGGAGGAGTCCTGGGTAACGGTAACAACACCAC
CGTCTTCGAAGTTCATAACACGTTCCCATTTGAAACCTTCCGGGAAGGACAGTTTCAGGTAGTCCGGGATGTCAGCCGGGTG
TTTAACGTAAGCTTTGGAACCGTACTGGAACTGCGGGGACAGGATGTCCCAAGCGAACGGCAGCGGACCACCTTTGGTAACT
TTCAGTTTAGCGGTCTGGGTACCTTCGTACGGACGACCTTCACCTTCACCTTCGATTTTCGAACTCGTGACCGTTAACGGAA
CCTTTCCATACATGACCATGTTCTCTCGTCTGATTAGCATCGTGAGCCTGATTCTGTCCTTCTACTTCGCTTACAAATACCG
TTATCGTGTGATTAACGCGGTGCTGGGCCGTCGCTGGCTGCGTAAAGTTATTATCGGTTTTGCCATGCAGATTCCGATGATT
CGTGACCGTATGCTGGGTAGCGTTCTGCAAAGTAACCGTCCGCAAAATGTGTAA 5'

Aroma module characterization

To characterize the aroma module, the process of sequencing was made too

3' TAAATAAAAAGTTTTTTCTAATGCGTTTCTTCTCCTACAACCGAAAACACCGGGTCAGTGAGCGAGGAACCTGCATAAC
GCGAAGCACGCTTTTCCGCAAGAAGAAAAAGGGCAGGGTGGTGACACCTTGCCCTTTTTTGCCGGACTGCAGCGGCCGCTAC
TAGTATTAGCGATCTACACTAGCACTATCAGCGTTATTAAGCACCGGTGGAGTGACTACCTTCAGCACGTTCGTACTGTTCA
ACGATGGTGTAGTCTTCGTTGTGGGAGGTGATGTCCAGTTTGATGTCGGTTTTGTAAGCACCCGGCAGCTGAACCGGTTTTT
TAGCCATGTAGGTGGTTTTAACTTCAGCGTCGTAGTGACCACCGTCTTTCAGTTTCAGACGCATTTTGATTTCACCTTTCAG
AGCACCGTCTTCCGGGTACATACGTTCGGTGGAAGCTTCCCAACCCATGGTTTTTTTCTGCATAACCGGACCGTCGGACGGG
AAGTTGGTACCACGCAGTTTAACTTTGTAGATGAACTCACCGTCTTGCAGGGAGGAGTCCTGGGTAACGGTAACAACACCAC
CGTCTTCGAAGTTCATAACACGTTCCCATTTGAAACCTTCCGGGAAGGACAGTTTCAGGTAGTCCGGGATGTCAGCCGGGTG
TTTAACGTAAGCTTTGGAACCGTACTGGAACTGCGGGGACAGGATGTCCCAAGCGAACGGCAGCGGACCACCTTTGGTAACT
TTCAGTTTAGCGGTCTGGGTACCTTCGTACGGACGACCTTCACCTTCACCTTCGATTTTCGAACTCGTGACCGTTAACGGAA
CCTTTCCATACATGACCATGTTCTCTCGTCTGATTAGCATCGTGAGCCTGATTCTGTCCTTCTACTTCGCTTACAAATACCG
TTATCGTGTGATTAACGCGGTGCTGGGCCGTCGCTGGCTGCGTAAAGTTATTATCGGTTTTGCCATGCAGATTCCGATGATT
CGTGACCGTATGCTGGGTAGCGTTCTGCAAAGTAACCGTCCGCAAAATGTGTAA 5'

Experiments- Return to the top

It is needed to characterize and prove the modules, here are the results of the experiments made in order to accomplish it.

Capture

Aroma

UV Experimentation - Return to the top

During 2 hours the four Petri dishes were exposed to UV irradiation. None of them showed any change.

Repetition UV Experimentation - Return to the top

Two Petri dishes inoculated by streak: One with NhaS Red and the other with NhaS White. Both exposed during 30 minutes to UV irradiation at 302 nm and none of them showed any change.

With no time exposed to UV irradiation:

After 30 minutes:

Viability test of the NhaS gene containing bacteria in salt [Capture Module] - Return to the top

Experiment #1

Bacteria transformed with the capture plasmid were inoculated in Petri dishes with different concentrations of salt

All the bacteria containing the NhaS in pSB1C3 (Red and White) survived to a 10% concentration of salt.

None of the control group lived in any concentration of salt.

The first time the experiment one was performed, bacteria transformed with the capture plasmid were inoculated in Petri dishes with different concentrations of salt, but this time the mayor concentration is higher (15%)

All the bacteria transformed with NhaS in pSB1C3 (Red and White) lived in the 15% saline medium.

All the control bacteria exposed to any concentration of salt died.

All the control bacteria inoculated only in LB medium (without salt) lived.

Experiment #2 - Return to the top

In the experiment #1, it was proved that the NhaS transformed bacteria (red and white) survived to a saline environment with LB agar. Then it order to know if it would survive only in a saline medium, it was designed a second experiment where the bacteria was inoculated in erlenmeyer flasks with only salty water at different concentrations (1%, 2.5% 5%, 10% and 15%). None of all the inoculated erlenmeyer flasks was murky, it means that all of the bacteria was dead.

Experiment # 3

AQUI D:

Aroma Qualitative Experiments [Aroma Module] - Return to the top

Experiment 1 - Test tubes

AQUI S;

Experiment 2 - Petri dishes

Random people were chosen to smell our bacteria, four people per each concentration. The experiment was performed with three different concentrations of salicylic acid, which were of 10 mM, 20 mM, and 30 mM. All of the samples contained salicylic acid. There was a controlled group grown bellow and above the 32 ºC and a group with transformed bacteria with the Aroma module, also grown bellow and above the 32 ºC; per each concentration.

Those are the words that people used to repeat, or synonyms of what the said, because all the opinions were described in a different way.

Qualitative Aroma experimentation in petri dishes