Cadmium/Heavy metals AND Gate

05/29/2012

Our group is in charge of building part of the “and” construction. We started analyzing if the plasmid we have with P4 actually had what we needed.

The plasmid PRMn25 contains the protein P4. It has Amp100 resistance and comes in Escherichia coli NFI. Cells were lysed to make sure the plasmid was present in these cells (5000bp). We ran a gel with 3 lysis, a sample from the plasmid PFRC54 (A3 promoter) and a sample of total DNA from the strain from which PFRC54 was obtained.

05/31/2012

Plasmid PRMn25 was digested with SpeI and EcoRI. We do not have the sequence of the plasmid, but there seem to be several restriction sites for SpeI and just one site for EcoRI. The digestions were left for 4 hours and they were plused in the microwave 3 times, 10 seconds each time. Only Dulce’s digestion worked, even though it was the “dirtiest” sample.

06/06/2012

We repeated the lysis of PRMn25.
Digestions (20 µl)
H2O 12 µl
Enzime 1 µl
Buffer 10x 2 µl
Plasmid 5 µl
37ºC

PRMn25 was digested with EcoRi and BamHI. Since we don’t have the sequence we used these enzyme to confirm the restriction sites mentioned in Rojo et al. (1990). These sites were corroborated and the vector was linearized. We recycled the gel used for the PRMn25 lysis, which is why the gel seems out of phase.

06/08/12

The lysis worked so we transformed PRMn25 in DH5. We performed a lysis on these cells to see if they had been transformed correctly with PRMn25.

1. 10kb ladder
2. PRMn25 (P4) lysis 1
3. Cell lysis of cells transformed with lysis 1
4.Cell lysis of cells transformed with lysis 1
5. Cell lysis of cells transformed with lysis 1
6. Cell lysis of cells transformed with lysis 1
7. Cell lysis of cells transformed with lysis 1
8. Cell lysis of cells transformed with lysis 1
9. Cell lysis of cells transformed with lysis 1
10. Cell lysis of cells transformed with lysis 1
11. Cell lysis of cells transformed with lysis 1
12. Cell lysis of cells transformed with lysis 1


We also transformed PFRC54. [TRANSFORMATION PROTOCOL]

06/11/12

We designed primers for our project. (Note from 07/10/12: we redesigned the primer for LasR, since the sequence was incorrect.)
OLIGOS 14/06/12
LASR
UPPER 5'-3'
PREFIX+RBS+SPACER+LASR

GTTTCTTCGAATTCGCGGCCGCTTCTAGAG AAAGGTGGTGAA TACTAG ATGGCATTAGTAGAT

LOWER 5'-3'

SUFIX+LASR

GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTA TCATAATGTAATTAA

P4 5'-3'

PREFIX+RBS+SPACER+P4

upper

GTTTCTTCGAATTCGCGGCCGCTTCTAGAG AAAGGTGGTGAA TACTAG ATGCCTAAAACACAA

SUFIX+P4

lower 5'-3'

GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTA CTACACCATACTTTT
A3 (PROMOTER)

UPPER 5'-3'

PREFIX+A3

5' GTTTCTTCGAATTCGCGGCCGCTTCTAGAG taactttttgcaaga 3'

LOWER 5'-3'

SUFIX+A3

5'GTTTCTTCCTGCAGCGGCCGCTACTAGTA ctacttaattatacc 3'

RFP

UPPER 5'-3'

PREFIX+RBS+SPACER+RFP

GTTTCTTCGAATTCGCGGCCGCTTCTAGAG AAAGGTGGTGAA TACTAG ATGGCTTCCTCCGAA

LOWER 5'-3'

SUFIX+RFP

GTTTCTTCCTGCAGCGGCCGCTACTAGTA TTATTAAGCACCGGT

GUSA

UPPER 5'-3'

PREFIX+RBS+SPACER+GUSA

GTTTCTTCGAATTCGCGGCCGCTTCTAGAG AAAGGTGGTGAA TACTAG atgttacgtcctgta

LOWER 5'-3'

SUFIX+GUSA

GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTA tcattgtttgcctcc

ARAC without LVA (version 2 registry part: BBa_C0080)

UPPER 5'-3'

PREFIX+RBS+SPACER+ARAC

GTTTCTTCGAATTCGCGGCCGCTTCTAGAG AAAGGTGGTGAA TACTAG ATGGCTGAAGCGCAA

LOWER 5'-3'

SUFIX+ARAC

GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTA CAACTTGACGGCTAC

06/14/2012

We obtained the terminator from the 2012 distribution (plate 2 24C) and we transformed the DNA in DH5. The terminator (BBa_B0014) comes in the PSBIAK3 plasmid with resistance to Amp and Km. The RFP (BBa_E1010) comes in the plasmid PSBI2K3 with resistance to Amp and we obtained it from the 2012 distribution plate 2 (17E). We obtained the purified plasmid of the RFP from the AraC AND team. (Diego, Jonathan, and Abiel).

1. 1 kb ladder.
2. BBa_B0014 (1) – terminator
3. BBa_B0014 (4) – terminator
4. BBa_E1010 (RFP) Lysis
5. purified BBa_E1010 PSB12K3 (AraC AND team)
6. 700 bp ladder

06/15/12

Due to the failed digestions, we did the RFP and terminator lysis again.

1. 1 kb ladder
2. Terminator lysis (BBa_B0014)
3. RFP lysis (BBa_E1010)
The AraC team has the terminator plasmid purified, we are thinking of using that one.