Team:Ecuador/Results

iGEM ECUADOR
` C-lastin, Interlab

RESULTS

Index

PHASE I: CONSTRUCTION OF THE COMPOSITE PARTS CBD cipA-BMP2 and CBD cipA-sfGFP
Experiment 3: Purification of the PCR products of the gBlocks

Lane 1: 1kb DNA ladder
Lane 6: Purified PCR product of CBD gBlock
Lane 7: Purified PCR product of BMP2 gBlock
Lane 8: Purified PCR product of sfGFP gBlock
Gel composition
Agarose gel 1%
1X TAE Buffer

Competent Cells
Experiment 5: Transformation protocol

EXPERIMENT 6: Extraction of plasmid DNA

Agarose gel 1%
1X TAE Buffer
Lane 1: High Mass DNA ladder
Lane 2: psb1c3-CBD cipA-sfGFP plasmid
Lane 3: psb1c3-CBD cipA-BMP2 plasmid

EXPERIMENT 7: Enzymatic digestion of the plasmids containing psb1c3 as backbone

Agarose gel 1%
1X TAE Buffer
Lane 1: High Mass DNA Ladder
Lane 2-4: Digested product of psb1c3-CBD cipA-BMP2.
Lane 5: Digested product of psb1c3-CBD cipA-sfGFP.

PHASE II: CONSTRUCTON OF THE COMPOSITE PART CBD cipA-ELP
Experiment 1: Amplification of ELP from pET-24a-ELP[V-150]

Agarose gel 1%
1X TAE Buffer
Lane 1: 100 pb DNA ladder
Lane 2: PCR ELP product from pET-24a-ELP[V-150] using the primers Forward 1 y Reverse1
Lane 3: PCR ELP product from pET-24a-ELP[V-150] using the primers Forward 1 y Reverse3
Lane 3: PCR ELP product from pET-24a-ELP[V-150] using the primers Forward 1 y Reverse2

Experiment 2: Domestication of the ELP550 amplicon

Agarose gel 1%
1X TAE Buffer
Lane 1: 100 pb DNA ladder
Lane 2: Purified ELP550
Lane 3: No sample
Lane 3: Unpurified ELP550

Phase III: Construction of the cellulose production module
Experiment 1: Amplification of LacI from pET-24a-ELP[V-150]

 

Agarose gel 1%
1X TAE Buffer
Lane A: 1 kb DNA ladder

Lane B: BscA 2536 bp
Lane C: BscB 2827 bp
Lane D: empty
Lane E: BscC2 2139 bp

 

Agarose gel at 1%
1X TAE Buffer
Lane A: 1 kb DNA ladder
Lane B: bscC1 2152 bp

 

Agarose gel 1%
1X TAE Buffer
Lane A: 1 kb DNA ladder

Lane B: BscA 2536 bp
Lane C: BscB 2827 bp
Lane D: empty
Lane E: BscC2 2139 bp



 

Agarose gel at 1%
1X TAE Buffer
Lane A: 100 pb DNA ladder
Lane B: unidentified sample
Lane C: BscD 849 pb
Lane D: BscD 849 pb

The parts to be assembled with psb1C3 were amplified using 8% DMSO due to its primers with high concentration of GC and the tendency to form secondary structures. As shown in Figures 1, 2 and 3, all the amplicons were of the expected length. The BscD was the only sequence that after the PCR still had a nonspecific amplicon, but its fluorescence was weaker than the fluorescence of the expected length band (figure 3).

 

Agarose gel at 1%
1X TAE Buffer
Lane A: 100 pb DNA ladder
Lane B: lineal Psb1C3 2135 bp
Lane C: lineal Psb1A3 2050 bp
Lane D: bsCmcax 1522 bp
Lane E:bsCcpax (1st attempt) 1512 bp
Lane F:bsCcpax (2nd attempt) 1512 bp
Lane G: bsCcpax (3st attempt) 1512 bp
Lane H: LacI 1420 bp

The parts to be assembled with psb1A3 were also amplified using 8% DMSO for the same reason explained above. As shown in Figure 4 all the amplicons were of the expected length. The BsCcpax PCR attempts gave us different quality amplicons using different annealing temperatures.

 

Agarose Gel 1%
1X TAE Buffer
Lane A: 1 kb DNA ladder
Lane B: gibson assembly psb1C3
Lane C: gibson assembly psb1A3

Using the Gibson assembly kit instructions from NEB we could not see any bands representing the expected ligation product for both plasmids (psb1C3 and psb1A3) (figure 5). A later attempt was done using DMSO, but no change was seen.

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