Team:Macquarie Australia/WetLab/Notebook

Welcome to our Notebook! This page has been created to detail our projects progress since its conception. Click on the section titles below to expand pages that contain information regarding what we worked on and achieved each week. For a more detailed summary of what we achieved please visit the Results page.

Week 1

Biobrick stocktake of 2013 iGEM Macquarie_Australia parts: 11/11/13

• ChlG - sufficient plasmid stock
• DVR1 - sufficient plasmid stock
• ChlM - sufficient plasmid stock
• ChlI2 - need more plasmid stock
• POR- need more plasmid stock
• YCF - need more plasmid stock
• Plasto - need more plasmid stock
• GUN4- need more plasmid stock
• CTH1 - need more plasmid stock
• ChlD - need more plasmid stock. Question whether the 2013 part is really the reported sequence - something appears to be missing.

Send all for re-sequencing to verify DNA sequence as per registry entries.
DVR1 re-tranformation: Was re-done using gibson assembly and then transformed.

Week 2

ChlD Fix: ChlD is missing 50 bp. Strategy to correct is to use ApaI and MluI restriction enzymes to cut out 50bp from clone of ChlD in pET vector from Willows group and re-insert into our BioBrick vector.

ApaI and MluI were used in a single digest according to manufacturer's instructions and as per ligation protocol on methods wiki. Fragments run on 1% agarose and gel purified. However, digestions were incomplete as viewed on agarose gel. Need to do separate digests for next attempt.

Double restriction enzyme digest was carried out to combine PCR1 and Gblock2. After the two sections were ligated and extended, straight PCR was done. The PCR worked as judged by agarose gel.

Week4

Biobrick (BB) ChlI1 is combined with ChlI2 biobrick in AMP backbone. Method is via 3A assembly. Use 500ng of each part and insert into 500ng of amp backbone. Ligation for 16oC for 30 mins then 80oC for 20 mins. Leave plates over weekend at room temperature.

Growth on plates : 1 colony on low plate, hundreds on high plate.

• G1 - G1F + G1R
• G2 - G2F+ G2R
• G3 - G3F + G4R
• G4 - G4F+ G4R
• G5 - G5F + G5R
• G6 - G6F + G6R
• PCR1+ G2- H1F+ G2R
• (PCR1 + G2) + G1
• G3 + PCR2- G3F+ H2R
• G5 + G6- G5F +G6R

Increase stocks : Did plasmid preps to get more of: ChlI1; ChlI2; YCF54; ChlP, DVR1; POR

Attempt to make ChlH (BBa_K1080001) using combination of gblocks and PCR products, as designed by Macquarie_Australia 2013 iGEM team.

Assembly strategy is: G-Block –1 (470bp) + PCR-1 (304bp) + G-Block-2 (499bp) + G-Block-3 (499bp) + PCR-2 (984bp) + G-Block-4 (500bp) + G-Block-5 (481bp) + PCR-3 (673bp)

Extremely faint bands are seen for

Another attempt to assemble chlD from PCR fragments

• G Block 1
• G Block 4
• G4 + (G5-G6)
• G1 + PCR1
• G2 + (G3 + PCR2)

Week 1

ChlM in AMP backbone vector and lac in backbone were digested using iGEM restriction digestion protocol EcoRI and Pst1 restriction enzymes.

Tuesday

10/12/13

The fragments to be PCR’d and the primers are presented on the following table;

Small amount of growth seen on ChlM, lacA & lacB indicating that they were successfully incorporated into the DHS← cells. Sequencing to confirm required

Transformation of Kanamycin Resistant backbone

We need more of the kanamycin biobrick. Transformation of kanamycin backbone into E. coli cells to produce large amounts of KAN backbone for future ligations.

Monday

09/12/13

PCR reaction for ChlH and ChlD

The overall of the aim of the week was to build ChlH fragment and PCR ChlD. Using the standard PCR protocol, G1+H1, G2 (G3+H2), G4 (G5+G6), ChlD (2) and ChlD (3) were run.

The result showed another G1+PCR1 failure. It was also suggested however to use BioBrick primers. Distinct bands for G2+(G3/PCR2) and G4+(G5/G6) were present and proved correct. This assumption was made that these results were correct.

ChlD 2 and 3 showed a band present at approximately 1500 bp which was also assumed to be correct in relation to the actual size of 1681 bp.

Figure2: The next step was to rePCR G1+PCR1 with BBF + HR2 and BBvF + HR2, gel extraction of G2+(G3/H2) and G4+(G5/G6) ,ChlD 2 and 3.

Tuesday

10/12/13

The fragments to be PCR’d and the primers are presented on the following table;

The standard PCR Method was adopted to run the reaction

Figure 3: All but ChlI1 and ChlI2 failed

Continued ChlH construction

At this stage, the ChlH gene construct was continued;

G1-P1-G2-G3-P2-G4-G5-G6

The ChlD gene was cut from the gel and extracted with another attempt to PCR.

To test for protein expression, the successful 3A gene was combined with lac creating a composite.

Continuing the construct of ChlH, a PCR reaction was performed to identify the successful or unsuccessful attempt in the composite build in addition to DVR1 identification.

Table 2: The PCR reaction screening attempting to construct chlH failed

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Donec sed metus est. Nullam ut enim urna. Sed sit amet bibendum velit. Morbi erat mauris, commodo at mi non, malesuada pharetra tortor. Nunc hendrerit nulla dignissim odio mattis congue. Fusce non magna sem. Vivamus aliquam feugiat leo sed porta. Mauris placerat non eros quis ornare. Proin viverra sodales ullamcorper. Mauris ac turpis eu risus efficitur ultricies id sit amet neque. Class aptent taciti sociosqu ad litora torquent per conubia nostra, per inceptos himenaeos. Donec bibendum arcu justo, lacinia cursus justo sodales eget. Mauris vitae augue gravida, bibendum metus sit amet, rutrum magna. Fusce sagittis leo iaculis varius interdum. Class aptent taciti sociosqu ad litora torquent per conubia nostra, per inceptos himenaeos.

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Donec sed metus est. Nullam ut enim urna. Sed sit amet bibendum velit. Morbi erat mauris, commodo at mi non, malesuada pharetra tortor. Nunc hendrerit nulla dignissim odio mattis congue. Fusce non magna sem. Vivamus aliquam feugiat leo sed porta. Mauris placerat non eros quis ornare. Proin viverra sodales ullamcorper. Mauris ac turpis eu risus efficitur ultricies id sit amet neque. Class aptent taciti sociosqu ad litora torquent per conubia nostra, per inceptos himenaeos. Donec bibendum arcu justo, lacinia cursus justo sodales eget. Mauris vitae augue gravida, bibendum metus sit amet, rutrum magna. Fusce sagittis leo iaculis varius interdum. Class aptent taciti sociosqu ad litora torquent per conubia nostra, per inceptos himenaeos.

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Donec sed metus est. Nullam ut enim urna. Sed sit amet bibendum velit. Morbi erat mauris, commodo at mi non, malesuada pharetra tortor. Nunc hendrerit nulla dignissim odio mattis congue. Fusce non magna sem. Vivamus aliquam feugiat leo sed porta. Mauris placerat non eros quis ornare. Proin viverra sodales ullamcorper. Mauris ac turpis eu risus efficitur ultricies id sit amet neque. Class aptent taciti sociosqu ad litora torquent per conubia nostra, per inceptos himenaeos. Donec bibendum arcu justo, lacinia cursus justo sodales eget. Mauris vitae augue gravida, bibendum metus sit amet, rutrum magna. Fusce sagittis leo iaculis varius interdum. Class aptent taciti sociosqu ad litora torquent per conubia nostra, per inceptos himenaeos.