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

Figure 4 chlH and DVR1 Gibson assembly gel

The next step was the insertion of DVR1 into the plasmid vector. The plasmid vector and the plasmid containing the gene of interest were ligated with EcoR1 and Pst1. The gene was introduced into the vector my means of 1 vector to 3 insert to maximise insertion efficiency.

ChlH construction by Gibson assembly

The failure of the construction of the ChlH gene subjected the attempt in the construction of the gene using Gibson assembly. The provided gel image proved the construction also failed.

Figure 5 PCR of ChlH Gibson Assembly

Tuesday

ChlH construct PCR

In the attempt to yield a positive result in the construction of ChlH, each P1+G2, (G3+P2) and (G4-G5-G6) were PCR’d separately in the attempt to successfully join the individual components.

10/12/13

Table 3 PCR of ChlH fragments

Thursday: 09/01/14

Gel analysis of PCR gel of ChlH constructs and ChlD

The results obtained would indicate the band to extract for Gibson assembly. The gel image showed positive results

The marked were cut out and stored for gel extraction. Figure 6 PCR gel analysis of ChlH constructs and ChlD. To compare the sizes, 25-500ng of plasmid were digested with and without lac. The expected size was approximately 200 bp. The amplification of ChlD was faint indicating an issue with the construction of the gene.

Friday

10/01/14

ChlH screening

The bands on gel corresponding to ChlH were extracted to screen for the correct sizes. The result of the gel extraction showed low concentration indicating poor construction of gene.

Figure 7 ChlD/ ChlI Plastocyanin screening: Digests were performed with enzymes EcoR1 and Pst1 to comment on the sizes of the inserts including ChlD, ChlI1 and Plastocyanin. These were also run against the corresponding components including lac.

Saturday

11/01/14

Table 4: Continued construction for ChlH PCR

Figure 8 The results obtained from the gel yielded a successful result for P1-G2, responsible for the construction of CHlH and negative results for the remaining samples on the gel.

Thursday

30/01/14

PCR: It is thought that the excess template in the previous PCR may have been responsible for the failure of PCR amplification. Template dilutions of 1/10 and 1/100 were tested by running another pcr.

The PCRs carried out were:

• ChlD (new template), diluted
• G3 -P2 PCR template
• ChlH gel run + extracted template

Figure 9 ChlD and ChlH G3-P2 PCR template did not work, however, ChlH G3 -P2 PCR template was successful.



PCR for ChlD blocks:

G4 + (G5-G6) X3 = G4F + G6R

P1- G2 (from the original templates) x3 = P1F +G2F.

Figure 10 G4 + (G5-G6) and P1- G2 PCRs worked. G4 + (G5-G6) showing a band of 1700 bp in length and P1-G2 showing 800 bp in length.



PCR continuation:

(P1-G2) + (G3-P2)

G3-P2 + G4-G5-G6

Figure 11 None of the PCRs from the ChlD blocks worked - clear, desired bands were not found

Week 1

Monday

3/2/2014

Digestion of DVR1 was run.

Following the digest, a ligase reaction was conducted and transformation performed. Note, the concentration of DVR part in comparison to the concentration of the plasmid was 1.5 times more. Plates incubated overnight.

Protein expression of lac+plasto & lac+ChlI

Expression of protein via lac promotor using 2uL of IPTG was done for each sample to amplify protein expression.SDS-PAGE was run according to methods.

Tuesday

4/2/14

SDS PAGE attempt #2

Here we conducted a second SDS PAGE for lac plasto and lac ChI1.

Lane order: 1-4 lac plasto, 5 is the ladder, 6-10 lac ChlI1

Expression of proteins was not visible by eye. No image of the gel was recorded. We think we need to do mass spec (MALDI/TOF/TOF) to identify proteins in bands. Discuss with APAF (Australian Proteomic Analysis Facility) at Macquarie University to ask if they can help us with performing mass spec.

Testing new ligase: New ligase purchased as concerns were that our ligase was old and the reason ligations were not successful

ChlH was digested with E+P restriction enzymes as per methods. To ligate, the ligation mixture comprised of 8.5uL DNA, 0.5uL ligase and 1uL of buffer.

Figure 12 The gel showed inconclusive results and requires further clarification. Further testing methods such as re-inserting the biobrick into another vector and growing it on a plate with the second vector antibiotic have been suggested.

PCR:

Fragment 1 - (P1-G2) + (G3-P2) = P1F, P2R

Fragment 2- (G3-P2) + (G4-G5-G6) = G3F, G6R

Fragment 3- (P1-G2) + (G3-P2) + (G4-G5-G6) = P1F, G6R.

For such large fragments, the preliminary melting step was completed twice prior to the addition of the primers because of the long fragments. The rest of the process was continued on the regular loop as in other PCR protocol.

Wednesday

5/2/14

Western Blot:

Western blot for ChlI1 and plasto were carried out.

Results: The plasto lanes did not show any expression, however ChlI showed good expression in lanes 2,4 and 5.

Figure 13 The gel showed inconclusive results and requires further clarification. Further testing methods such as re-inserting the biobrick into another vector and growing it on a plate with the second vector antibiotic have been suggested.

Thursday 6/2/14: Gibson Assembly of ChlH:

G1: 3uL

P1-G2 exosap: 0.5uL

G3-P2 exosap: 4.8uL

G4-G5-G6 exosap: 1.0uL

Cam vector: 29uL with Gibson mix: 12.3uL or AMP vector 1.4uL with Gibson mix: 10.8 uL

- Plated out

PCR of Gibson Assembly product for ChlH: Standed PCR x2 using BBF/ BBR/ BBVF2, BBVR.

Results:

Week 2

Wednesday

12/2/14

Nanodrop of ChlH fragments

ChlH Fragments:

P1-G2:

• A= 19.3 ng/ml

• B= 23.4 ng/m

• C= 18.2 ng/m

G3-P2:

• A = 27.8 A= 141ng/ml

• B= 9.9 ng/ml

• C= 47.5 ng/ml

G4-G5-G6:

• A= 141ng/ml

• B= 24.6 ng/ml

• C= 62.5 ng/ml

ChlD Fragments: D1, D2, D3

Thursday

13/2/14

Gel Electrophoresis for ChlH and ChlD fragments:

Top Gel Lane order: 1- Ladder, 2- G1, 3-5 - P1-G2, 6-8- G3-P2, 9-11- G4-G5-G6, 12-14- ChlD

Bottom Gel Lane Order: 1+ 2- Ladder, 3-5- P1-G2, 6-8- G3-P2, 9-11 G4-G5-G6

Results: ChlH fragments appear not have been digested. P1-G2 and G4-G5-G6 didn’t have plasmids on the gel so they did not digest. ChlD has digested with MLU and partially APAI. ChlD plasmids from lanes 12 and 13 were added together for further re-digestion.

New Digestion using E+P from previous gel electrophoresis for ChlH:

Lane Order: 1- G1, 2- G3-P2 A, 3- G3-P2 B, 4- G3-P2 C, 5- G4-G5-G6 A, 6- G4-G5-G6 C, 7- Plasto Control

New Digest for ChlD with APAI: The ChlD being redigested is a combination of lanes 12 and 13 from the previous electrophoresis gel.

ChlD Ligation : ChlD was ligased and transformed into E.coli. 2 colonies grew on the 300uL plate and 1 colony on the 30uL plate.

Friday 14/2/14

Plasmid nanodrop

Lac	Gun	A	33.8 ng/µl
		B	17.8 ng/µl
Lac	ChlM	A	33.5 ng/µl
		B	26.8 ng/µl
Lac	ChlP	A	32.2 ng/µl
		B	14.4 ng/µl
Lac	ChlI2	A	18.5 ng/µl
		B	15.2 ng/µl
Lac	POR	A	35.1 ng/µl
		B	24.5 ng/µl
Lac	ChlG	A	8.3 ng/µl
		B	13.2 ng/µl
Lac	YCF54	A	29.3 ng/µl
		B	33.9 ng/µl
Lac	CTH1	A	59.1 ng/µl
		B	51.6 ng/µl

Table 6 ; : Biobrick concentrations

ChlH re-digest:

Digests checking biobricks:

With/without lac	Biobrick	Fragment	Expected weight	Measured
Lac	Gun	A	930	~930
		B		~930
Lac	ChlM	A	873	~1050
		B		~1050
Lac	ChlP	A	1299	~1500
		B		~1500
Lac	ChlI2	A	1212	~1400
		B		~1250
Lac	POR	A	1067	~1250
		B		~1250
Lac	ChlG	A	1050	~1250
		B		~1250
Lac	YCF54	A	471	~650
		B		~650
Lac	CTH1	A	1152	~1350
		B		~1350
	DVR1	A		~1106
		B		~1106
		C		~1106
		D		~1106

Results: Majority look as though they match the expected band length. All digests excluding DVR1 include lac.