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Notebook

Semester 1

Preliminary work – Lab setup, training, etc.

In Semester 1, we began the process of recruiting the team and setting up the lab. Because our lab was new, we needed to order all supplies, secure scientific sponsorships, and establish protocols. The founding team was also able to do some exploratory lab work before the changeover to the full team later in the semester.
Lab members were recruited from the wider University of Melbourne student community through an advertising campaign, followed by a selection process. The team underwent training during the Easter break and the following weeks.

 

Week beginning 6 January

Grew stock of competent DH5α E. coli. Cells stored at -80°C.

 

Week beginning 13 January

Note that the 2014 founding team originally came up with the concept for a star peptide similar to the USP peptide reported on the Project page. This peptide was meant to be unstructured. It contained an RGD motif to support potential binding of the peptide to integrin (hence why it was termed the RGD peptide). Unlike the peptides reported on the project page, the RGD contained a TEV protease recognition sequence. This would have allowed it to be co-expressed with the TEV protease, and therefore cleaved to form a star peptide inside the cell.

The RGD peptide sequence was: MRV LLF LLL SLF MLP AFS RGD RGD GGG AKQ RGG C GGR QKA GGG RGD RGD EQLYFQG RGD RGD GGG AKQ RGG C GGR QKA GGG RGD RGD HHHHHH

The founding team ordered this peptide's gene to be synthesised and most of the early work in the lab was focused on using it to practice lab techniques.

Week beginning 20 January

 (20-01-14)
RGD construct DNA arrived.
Completed transformation of DH5α competent cells with heat shock and LB (see Protocols).
OD of competent cell growth:

Digestion was performed using EcoRI (see Protocols).
Amount of plasmid was measured.
RGD001 – 37.1 ug/uL
RGD002 – 51.2 ug/uL
RGD003 – 34.5 uG/uL
Gel was run. No DNA was visible but the ladder was fine.
(24-01-14)

• Redid miniprep. Quantified RGD plasmid again.

RGD001 – 37.1 ug/uL
RGD002 – 51.2 ug/uL
RGD003 – 34.5 uG/uL

• Performed double digest of RGD plasmid with EcoRI and PstI (see Protocols).
• Performed gel electrophoresis of digested plasmid and undigested plasmid. Plasmids were present at approx 3.5 kb. RGD001 seemed to be the better digested plasmid and best to use for transformation.

 

Week beginning 27 January

• Transformed BL21 with RGD. Placed in incubator overnight at 37°C.
• Ran third gel of transformed DH5α (with RGD).
• Performed double digests before running another gel to check success.

 

Week beginning 3 February

Performed protein induction on cells containing RGD plasmid.
NOTE:

• Grew 800 mL culture and measured OD (absorbance at 600nm):

0 hrs – 0.184
1 hr – 0.322
2 hrs – 1.265
2.5 hrs – 1.471

• Despite overshooting OD target, went ahead with IPTG induction anyway.

 

Week beginning 10 February

Project design/planning
Did some project planning based on meeting with advisor Neil O’Brien-Simpson:
Selenocysteine
First thought when he saw the project was the liability of the disulfide bond (i.e. tends to fall apart under producing environments).
He suggested trying selenocysteine, because selenocysteine bonds are stronger and the amino acids should be sitting around in E. coli ready to use.
Comments on AMP
Suggested a good idea to stick with the Wiradharma, 2012 structure rather than alter too much so as to make comparisons easier.
We briefly discussed the mechanism of action of AMPs. As we know, there are several models, including the barrel stave, toroidal poor, and carpet models. In addition, peptide aggregation at the surface of the bacterial cell can lead to membrane thinning, which facilitates pore formation.
Neil wasn’t sure how the star polymers would work in particular. They may work using the above mechanisms… The disulfide bond may also be broken as the stars enter the cell cytoplasm.
Related work
A researcher by the name of Tam has developed a Multiple Antigenic Peptide (MAP) similar to ours. This peptide includes branched lysine residues. However, the problem with this molecule is that it is very challenging to synthetically produce.

 

Week beginning 17 February

• Began new protein expression method (see Protocols).
• Pelleted cells (see Protocols).

 

Week beginning 24 February

• Transformed DH5α Kan.R resistance (see Protocols). Placed 4 plates on incubator overnight.
• Transferred cells to 4°C. Colonies grew, suggesting the transformation was successful.

 

Week beginning 3 March

(04-03-14)

• To check success of transformation, we ran an SDS-PAGE gel at 150V, 400mA, 45 mins with positive controls and RGD protein (see Protocols):

• Transformed DH5α colonies with Magainin I.
• DH5α (MagI) transformed cells were miniprepped (see Protocols). Some of cell pellet was loaded into SDS-PAGE gel with two positive controls for His-tags. Results appeared successful so we continued onto Ni-NTA column purification.
• Unfortunately, Ni-NTA column purification failed.

 

Week beginning 10 March

Redid process from last week to try to diagnose what went wrong.
Double digestion of DH5α (Magainin I) colony miniprep products with EcoRI and PstII.
Made and ran DNA gel with double digest at 120V, 100mA, 35 minutes then 135V, 100mA, 15 minutes then 120V 3 minutes.

Results were visible for lanes 1 – 4, but digests were not visible.

 

Week beginning 17 March

(Recruitment of team members)

Week beginning 24 March

(Recruitment of team members)

Week beginning 31 March

(Recruitment of team members)

Week beginning 7 April

(Recruitment of team members)

Week beginning 14 April

(Recruitment of team members)

Week beginning 21 April

(Lab introductory training)

Week beginning 28 April

(Lab setup work)

 

Week beginning 5 May

Despite negative results on gel of double digest (thought to be due to faulty gel electrophoresis process), we went ahead with protein expression.
We also transformed BL21 with pMK-RGD vector (see Protocols) and ran a protein gel with pGEX, pET, pMK RGD and pMK Mag 1 (samples taken from the training week during the break).

Conducted miniprep of DH5α pMK-RGD culture and ran DNA gel on the RGD plasmid extracted.

 

Week beginning 12 May

Started protein expression on transformed BL21 pMK-RGD.

 

Week beginning 19 May

Completed protein expression, but cultures failed to grow.
Ran PCR for pMK RGD (reaction volume = 50 uL) with the following new recipe:

• 10X Thermopod Reaction Buffer – 5 uL
• 10 mM dNTP mixture – 1 uL
• 10 uM forward primer – 1 uL
• 10 uM reverse primer – 1 uL
• Template DNA – 1 uL
• Taq DNA polymerase – 0.5 uL
• Nuclease-free h3O – 40.5 uL

Conditions for the PCR cycle:

• 95°C – 2 minutes
• 30 cycles:
• 95°C – 20 seconds
• 60°C – 20 seconds
• 72°C – 20 seconds
• 72°C – 5 minutes
• 4°C – infinity (hold)

Performed protein induction on PMK-MAG1 .

Created a glycerol stock of BL21-RGD and BL21-MAG1 and stored it at -86°C.

 

Week beginning 26 May

(Exam period – no lab preparation work)

 

Week beginning 2 June

(Exam period – no lab preparation work)

 

Week beginning 9 June

(Exam period – no lab preparation work)

 

Week beginning 16 June

(Exam period – no lab preparation work)

 

 

Semester 2

Main project work begins.
With the lab set up, the team fully formed, and preliminary work completed, we began work on the project. ­Our focus throughout Semester 2 was on the design of the BioBricks, and ensuring that each of our proteins could be properly expressed in transformed bacteria after cloning and subcloning.

 

Week 1 – 23 June

(24-06-14)

• Plated BL21(DE3) + Magainin 1 glycerol stock on one plate using ‘spread’ technique at 12:45pm, placed in 37 degree incubator at 3pm (should be taken out at 11am next day [+20 hours]).

However, the plates had way too much growth, almost resembling a lawn, and couldn’t be used due to a need for ‘isolated’ colonies. After checking lab-book again, realised the glycerol stock was made using culture from already plated colonies and instead of just transformed cells.
New glycerol stock was used to plate again, this time using the ‘streak’ technique.

• SDS-PAGE gel with MagI pellet was run. 2 samples were prepared using approximately 100μL of SDS-PAGE Sample Buffer and a small amount of pellet. The samples were prepared using Ash’s method of quickly boiling the samples (placing tubes in heat block set at 100(99.9) degrees) to lyse the cells. Unfortunately, the samples thickened and SDS-PAGE could not be run. This may be a result of specific faults in the buffer recipe, so possibly use a RIPA buffer next time.

 

Week 2 – 30 June

Project design/planning
The team agreed to have a multi-track research program. What we are essentially doing at the moment is trying to attack this problem of synthesizing disulfide bonded structures from multiple fronts.
Tobias suggested a new concept for ligating peptides, carbohydrates, lipids and other biomolecules to the star peptide structure through native chemical ligation.

 

Week 3– 7 July

Project design/planning
Further work on the idea of native chemical ligation (NCL).
Summary of progress on shuttling the MAG 1 insert with the periplasmic export tag to the standard iGEM plasmid –

• Ran PCR on pMK-RGD using primers to make RGD compatible with pGEX vector
• Ran PCR product on gel
• Cut out PCR product from gel and use gel purification kit
• Ran restriction digestion on PCR insert product of 3a using BamHI and EcoRI
• Ran PCR purification using Thermo kit on product of 4a
• Transformed pGEX vector donated by a neighboring lab into DH5a, plate and incubated overnight
• Miniprepped plasmid
• Ran restriction digestion on plasmid product of 2b using BamHI and EcoRI

Summary of previous progress on inserting RGD coding region into pGEX vector, and expression in BL21(DE3) cells –

• Ran PCR on pMK-RGD using primers to make RGD compatible with pGEX vector
• Ran PCR product on gel
• Cut out PCR product from gel and use gel purification kit
• Ran restriction digestion on PCR insert product of 3a using BamHI and EcoRI
• Ran PCR purification using Thermo kit on product of 4a
• Transformed pGEX vector donated by a neighboring lab into DH5a, plate and incubated overnight
• Miniprepped plasmid
• Ran restriction digestion on plasmid product of 2b using BamHI and EcoRI

Decided to divide work into streams as previously planned – indicated in lab book as:

• pSB1C3-MAG1 subcloning
• pGEX-RGD cloning
• Cytoplasmic Star Magainin 1 cloning

 

Week 4 – 14 July

pSB1C3-MAG1 subcloning –

• Ran PCR on pMK-MAG1 using primers to remove TorTSS and add sticky ends for pSB (BBa_K525998). When run on a gel, the PCR product was a very clear band, indicating success.
• Ran restriction digestion on pMK-MAG1 PCR insert product using SpeI and PstI.
• Ran PCR on MAG1 using primers to remove TorTSS and add sticky ends for pSB (BBa_K525998) for a second time.

Cytoplasmic Star Magainin 1 cloning – Plated DH5a cells.

 

Week 5 – 21 July

pSB1C3-MAG1 subcloning –
We transformed DH5a with the pSB1C3 (RFP) plasmid provided by iGEM. We then miniprepped the cells. However, the nanodrop suggested that the concentration of the cells was extremely low. Gayle then ligated pSB1C3 with a MAG 1 insert. Because the concentrations were too low to see on a DNA gel, she re-transformed the plasmid to DH5a. The idea is that we should now be able to re-miniprep this DH5a and reclaim the ligated plasmid.
Cytoplasmic Star Magainin 1 cloning –
Did restriction digestion using PstI and SpeI on miniprepped T7 promoter containing pSB1C3. Ran the product on a gel, but it came up blank. We had a thorough discussion with Ash and David about why this could be. Ash suggested trying digesting with one enzyme at a time, having several lanes, each with different enzyme combinations.
A Google search suggested that there could be a problem with the miniprep procedure. Specifically, there could be some enzymes being left over from the miniprep which, when the samples were heated during the restriction digestion, ate up the DNA.
Therefore, Stan designed an experiment to test this. We had 5 conditions:

• the PSB1C3 plasmid directly after the miniprep, with no incubation or restriction digestion;
• the plasmid heated to 37° with restriction enzymes,
• the plasmid heated to 37° WITHOUT restriction enzymes,
• a PCR insert meant to go into the plasmid heated to 37° with restriction enzymes (the same restriction enzymes which would allow us to like it that insert into the pSB1C3 plasmid),
• the PCR insert heated to 37° without restriction enzymes.

So, we're testing the effect of heat and the effect of the restriction enzymes.
In the resulting gel, every lane was visible, except for the plasmid which had been double digested. This suggests that there was not anything wrong with the restriction enzymes per se, neither with the buffer nor the heating process. It argues against the hypothesis that there was some DNA degrading enzyme present after the miniprep could have been activated by heat.

 

Week 6 – 28 July

Cytoplasmic Star Magainin 1 cloning –
Thawed out the plasmid samples in sample buffer from yesterday and loaded them onto a 16 well agarose gel. Every lane was visible except for the Axygen miniprepped plasmid treated with SpeI. However, the Axygen miniprepped plasmid treated with a double digestion of SpeI and PstI DID show upon the gel. After speaking with David, he suspects that we perhaps neglected to put DNA into that well.
Note that these results are in direct contrast to the gel described earlier, where we had an identical plasmid (pSB1C3 containing the P7 promoter), picked from the same plate, using the same miniprep kit, same restriction enzymes (PstI and SpeI), for the same incubation period (2.5-3 hrs). That is, we did exactly the same thing, and this time, it showed up on the gel, whereas before it disappeared.
Given that the double digest with SpeI and PstI and EcoR1 and PstI worked, we went ahead and began culturing in 10 mL colonies from the same pSB1C3 plate used in the experiment above. We made 6, 10 mL cultures. To provide a backup, we also picked three colonies which had previously been plated out by Lumi containing GFP, RFP, or YFP, creating 3, 10 mL cultures which may also be miniprepped.
Further progress:

• Miniprepped 10 mL cultures of pSB containing the p7 promoter. Ran various restriction digests on the miniprep product in preparation for ligation of pSB with our MAG1 PCR insert.
• Ran the restriction digests from yesterday.  Results are very mysterious. Thus, re-ran restriction digests. Results favourably suggest one of the minipreps was successful.
• Gel extracted pSB1C3 digested with both SpeI and PstI.

 

Week 7– 4 August

pSB1C3-MAG1 subcloning – Transformation of pSB1C3 to DH5α (see Protocols).
pGEX-RGD cloning – Transformation of pGEX to DH5α (see Protocols).
Cytoplasmic Star Magainin 1 cloning –
We were originally planning to do the ligation reaction with the SpeI and PstI restriction enzymes AND the PCR insert. However, during that restriction digest of the PCR insert, DNA loading dye was erroneously added. We attempted to gel purify the PCR insert to remove the loading dye. However, for some reason the "PCR insert" ran at a very high molecular weight (approximately 2 kDa). Moreover, there is a strange double band.
Because the PCR insert appears to be screwed up, we will have to start from the PCR step again. Planning on ordering new primers which will allow the Magainin 1 insert to be biobrick compatible.

 

Week 8 – 11 August

pSB1C3-MAG1 subcloning –

• PLASMID:
• Stanislav performed a double restriction digest on the linearized plasmid backbone pSB1C3.
• Performed a miniprep on the 10 mL cultures. Confirmed the success of the miniprep/presence of the plasmid using a DNA gel.
• INSERT:
• Performed a restriction digestion on previous pMK-MAG1 plasmid stock in order to liberate the MAG 1 insert in preparation for ligation with pSB1C3 and ran a gel on the products. In this gel, a distinct band was observed corresponding to the approximately 2 kDa vector (pMK). However, the 6 kDa insert was not visible.
• In order to get more of the insert, we propagated more pMK-MAG1 plasmid.

pGEX-RGD cloning –

• Transformed DH5a competent cells with pGEX-6P3 and performed a miniprep to increase the amount of plasmid stock available for ligation. A DNA gel performed on the products suggested the miniprep was successful.
• A gel performed on PCR products of the RGD insert amplified with restrictions sites added for subcloning into pGEX gave odd results.

Both the PCR products created yesterday and the products done over the summer break showed over 10 bands in each lane. This suggests that there was nonspecific binding, leading to a variety of different molecular weight products. Spoke to advisor Ash who prescribed a new PCR temperature/timing cycle to minimise this problem.

Ran the PCR reaction (using Ash’s suggested solution) product on a gel. Increasing the annealing temperature in the PCR cycle did decrease the presence of multiple (~10)  bands. However, it also led to a general reduction in the intensity of the PCR product's signal on the gel. It's not clear that the changes to the PCR protocol actually reduced nonspecific binding so much as just decreased the reaction yield.

As suggested by our supervisor, Cheng, we reran the PCR reaction. This time, in order to eliminate the possibility of nonspecific binding, we cut out the RGD insert from the PMK vector using restriction digestion, gel extract the insert, and then used this insert as the PCR template.

Cytoplasmic Star Magainin 1 cloning –
We had previously ordered the primers which would both remove the periplasmic export tag and allow for ligation into Biobrick BBa_K525998 of our Magainin 1 Star Peptide insert. The problem with these primers is, however, that after the ligation, there would be the Spe1 restriction site at the 5' end of the protein coding region. Therefore, new primers were designed which would eliminate this problem. The protein coding region will be inserted after the T7 promoter in BBa_K525998. At the 5' end of the insert, XbaI will be used to join the insert to the plasmid's SpeI site. At the 3' end of the insert, PstI will be used to join the insert to the plasmids PstI site.

 

Week 9 – 18 August

pSB1C3-MAG1 subcloning –

• INSERT:
• Did a double digestion on the pMK-MAG1 plasmid.
• Ran the double digestion product on a DNA gel and gel-extracted.
• Ran an analytical amount of the digested linearized PSB1C3 backbone on a DNA gel. The plasmid was clearly visible on the gel (Gel C35).
• PLASMID: Did a double digestion on a significant amount of the linearized plasmid backbone and ran the double digestion product of pMK-MAG1 on a DNA gel. However, the results on the DNA gel (Gel C35) were inexplicable. We observed a smear in the lanes containing pMK-MAG1, not the clear bands we expected. Moreover, the insert was not visible at all in the lower regions of the gel.

Due to the anomalous results, we designed an experiment performing a series of restriction digestions on the pMK plasmid containing the Magainin 1 insert. Several controls, including single digestions and digestions using newer tubes of restriction enzymes EcoRI and PstI were performed.
pGEX-RGD cloning –
It was discovered that there was a mistake in the PCR primers we have been using up to this date. Specifically, the PCR primers amplified a segment of the DNA including the periplasmic export tag preceding the RGD protein coding region. Therefore, new primers had to be designed for PCR on the RGD insert.
Nonetheless, we did a double digestion on the pMK-RGD plasmid, making sure that the volume of the reaction is quite large so that the insert band was be clearly visible on the DNA gel. This measure is a backup in the event that the PCR primers are not specific in their binding to the template. If the primers are non-specific, then we will use the isolated insert as the template.

• PLASMID: Ran an analytical amount of the gel-extracted digested pGEX vector on a gel (Gel C35) and confirmed successfμL gel-extraction.
• INSERT: Ran the restriction digestion product (PMK-RGD) on a DNA gel. However, strangely we did not get the results we expected. Instead of seeing two bands, one corresponding to the vector in one to the insert, we saw approximately 5 bands. This suggests that the restriction digestion failed, and we might be seeing a multiple bands associated with the undigested plasmid.

Cytoplasmic Star Magainin 1 cloning –
Completed the PCR reaction to amplify the plasmid in preparation for ligation and ran on a gel. We have previously digested the destination plasmid using the relevant restriction enzymes. We did one final check to ensure that the gel extraction worked by running a sample of the product on another gel.
A double digestion was completed on a portion of the gel extracted PCR insert using XbaI and PstI, two restrictions sites which will allow us to ligate this insert with the pSB1C3 plasmid (containing the T7 promoter) at the biobrick suffix. The PCR insert was then PCR purified.
A ligation procedure was performed between pSB1C3 in the PCR insert. The ligation product was transformed to DH5a.

 

Week 10 – 25 August

pSB1C3-MAG1 subcloning –
A gel run on the restriction digestion experiment last week revealed that there was something wrong with our old EcoRI/PstI when used in combination.
We designed an experiment using gel electrophoresis of a combination of different enzymes, used on the same plasmid, repeated with our old and new enzymes. We found that with the NEW enzymes, the double digestion works, and with the old enzymes it doesn't work.
Strangely enough, it looks like the single digests with the old enzymes still work. It's as if there's some interaction effect when the old Eco and Post are used together where things go bonkers.
This experiment has also revealed a limitation of our cloning strategy. It turns out that there is actually a PstI cutting site in pMK. Hence, you will see that there actually more than two bands in the double digestions (and if you look closely, two bands in the single digestions with PstI). All is not lost, however, as we can still double digest pMK-MAG1 using different restriction enzymes, bypassing PstI. We can use EcoRI and SpeI, and this should work (there are no EcoRI or SpeI sites in pMK).
This sub cloning experiment was started from the restriction digestion point again. This time, however, we digested the destination vector (pSB1C3) and insert (contained in pMK-MAG1) with the NEW tubes of EcoRI and PstI. A gel was run on these products. Both the destination vector, pSB1C3, and the gel isolated insert were clearly visible on the gel.
A ligation reaction between the Mag1 insert and the pSB1C3 linearized vector was carried out. The ligation product was transformed to DH5α and plated to chloramphenicol-containing plates.
pGEX-RGD cloning – A gel was run on the PCR product (previously carried out). However, it appeared that the PCR reaction was inefficient; the product had a very feint band on the gel. After gel extraction of the product, nanodrop measurements suggested that the concentration was in the range 5-10 ng/uL. Because the ultimate goal of this PCR reaction was to create a lot of insert for a ligation later on, small amounts of insert DNA were not acceptable.
A second PCR reaction was run in order to hopefully increase the yield of the insert. The number of cycles was increased to 35 from 30. In a second condition, the gel extracted PCR product from the last reaction was used as the template.
The products of the PCR reaction of the RGD insert were run on a gel and were clearly visible at the correct molecular weight.
A restriction digestion of both pGEX-6P-3 and the PCR-amplified RGD insert was carried out. EcoRI and BamHI were used on both vector and insert to create complementary sticky ends. The reaction was completed and the products placed in the -20 freezer.
Cytoplasmic Star Magainin 1 cloning – Minipreps were performed on colonies containing the ligation and the products were run on a DNA gel (Gel C43). However, no DNA was visible on the gel, suggesting that the minipreps had failed. This is likely because the culture conditions of the miniprep were suboptimal. However, another gel showed that the PCR purified insert ran clearly on the gel. Thus, we can combine that insert with the vector and likely produce a clone again.
The ligation of the destination vector in the PCR amplified insert was repeated today. The ligation product was then transformed to DH5α. The ligation reaction to get the Magainin 1 insert into pSB1C3 was found to be successful. The MAG 1 insert was mixed with the destination vector pSB1C3 in a ligation reaction. The ligated product was transformed to DH5α and plated overnight. Colonies were picked from agar plate and then placed in a PCR reaction (PCR-based colony screening). The primers used in the PCR were specific to the Magainin 1 insert.
Performing a PCR on the colonies led to a propagation of the insert. This suggests that the colonies contained the Magainin 1 insert. And, because the colonies were chloramphenicol resistant, they should also contain the pSB1C3 vector. Thus, it appears that we have the vector with the insert!

 

Week 11 – 1 September

pSB1C3-MAG1 subcloning – From the plate of DH5α transformed with the ligation product, PCR colony screening was performed. Only one colony showed successful transformation on a DNA gel. This culture later showed no growth, and transformation was suspected to have failed. Because of this, a second transformation was carried out using the ligation product used for the previous transformation/plating, and appeared to be successful. Glycerol stocks were made of the cultures from the second transformation.
Later, more colonies were miniprepped, double digested, and ran on a gel. Strangely, as in previous gels, the only thing visible on the gel was a very faint band with molecular weight greater than 3000. It is unclear what that band corresponds to. Because the colony screening failed in this manner across many colonies and two different transformations, it suggests that there something went wrong during the ligation reaction or earlier.
pGEX-RGD cloning –
The insert for the pGEX RGD stream was PCR purified and then run on a gel. For unknown reasons, the PCR purification product was not visible on the gel. The nanodrop suggested that the concentration was 15 ng/µL. 10 µL of the product were run on the gel. It therefore should have been visible.
Note, however, that we observed a strange phenomenon where the DNA/loading dye was floating out of the well once loaded. After trying to load two wells, we eventually increased the loading dye amount to 5 µL. It didn't seem as if the DNA was floating out of the well this time, but it may still have happened.
The final, restriction digested, PCR purified insert was again run on a gel. This time, the gel was run with 25 µL of the insert. Inexplicably, on the gel we observed a very high molecular weight band (greater than 3000 kDa). This could possibly be contamination from the pGEX vector. In any case, the insert is not pure or visible on the gel, which suggests that we need to go back several steps and regenerate it.
Cytoplasmic Star Magainin 1 cloning – Made glycerol stocks of several cultures containing colonies which contain the correct clone (as verified by the colony screening). Miniprepped the cultures, double digested the resulting plasmid with EcoRI and PstI, and ran the product on a gel (A90). Fortunately, most of our plasmids were shown to have been successfully digested such that the vector and the insert were both visible as two distinct bands at the correct molecular weight on the gel. The plasmids were transformed to BL21(DE3) cells and SHuffle T7 cells.
Bacterial colonies were visible on chloramphenicol containing agar plates with the transformation product. Thus, it appears that this plasmid is now ready for protein expression.

 

Week 12 – 8 September

This week, agar swabs of bacteria containing three versions of the SUMO protease arrived from TU Delft. These bacteria were immediately plated out onto agar plates with no antibiotics. They were also plated onto plates with the antibiotics corresponding to the resistance of each plasmid.
Work on Delft’s SUMO protease is indicated by the stream label ‘Delft’.
pGEX-RGD cloning – Performed a double digest on the remaining sample of the PCR purified PCR product. However, when the entirety of this restriction digest was run on a gel, it mysteriously disappeared. This may be a problem with our apparatus, which has been acting up lately.
The PCR reaction to amplify the insert was carried out. Because we've experienced very low yields from previous PCR reactions (of the scale of 40 ng/uL), this time we increased the cycles from 35 to 45. Also, we used the PCR product from last time as the template. The reaction appeared to complete successfully and we ran the products on a DNA gel,  followed by a double digest with EcoRI and PstI. However, the gel was anomalous. Although the raw PCR product and the first PCR purification product were visible, the product of the restriction digest disappeared. This could possibly be due to Star activity, and appropriate follow-up experiments need to be run.
Cytoplasmic Star Magainin 1 cloning –
Protein expression turned out to be complicated when we discovered that a chloramphenicol-containing petri dish with a sham transformation showed colony growth. These colonies should not have appeared on the plate because the plate had this antibiotic resistance and BL21(DE3) does not inherently have resistance to chloramphenicol.
In order to troubleshoot, an experiment was set up with several controls, including conditions where a new chloramphenicol stock solution was made up and used, as well as conditions where different cell lines, separate from BL21(DE3), were plated out.
We discovered that the particular substrain of BL21(DE3) that we're using, BL21(DE3)_pLysS, is chloramphenicol resistant. The resistance comes from the pLysS plasmid. The pLysS is supposed to inhibit constitutive expression of the T7 polymerase. This is better for expression of toxic proteins, because it stops leaky expression of the toxic gene (which is why we chose this strain).
We confirmed this with a massive experiment with 12 controls. DH5a cells did not grow on the chloramphenicol plates, but did grow on agar plates with no antibiotic
Our BL21(DE3) cells grew happily on the chloramphenicol plates, even though there was no plasmid in them. The cells did not grow on ampicillin plates.
Fortunately, our competent SHuffle T7 cells are not resistant to chloramphenicol. Therefore, we proceeded with protein expression (see Protocols).
We then discovered there was an error in the transformation, in that an incorrect plasmid had been transformed.
Also, sequencing data for two of the colonies came back. Unfortunately, the sequencing reaction failed to produce a sufficiently long sequence. The primer/plasmid concentrations will be optimised for the next run.
TU Delft –
Today, all of the plates without antibiotics showed proliferative growth. Only one out of three plates with antibiotics showed growth. This was the gene for pBAD-UlpI-HIS6-TT. Several colonies from this plate were picked and cultured in 6 mL of LB overnight in preparation for plasmid miniprep.
For the remaining plates, colonies were picked and streaked onto antibiotic-containing plates to hopefully get some growth. Colonies which were plated to antibiotic-containing plates showed growth on these new plates.
Last weekend, the FG gene/MAP peptide gene had been transformed to SHuffle cells and plated onto chloramphenicol-containing plates. This week, protein expression was carried out.

 

Week 13 – 15 September

Continuing to attempt to subclone the RGD gene to both pGEX-6P3 and pSB1C3. Discovered that the reason our small ~300 bp PCR fragments were not visible was because we were using a 1% gel, 75 V run. We found that we should have been using a 2% gel at 150 V.
We optimized our PCR reaction for the RGDàpGEX cloning task. We used 45 cycles and the product of the previous reaction as the template.
Over the weekend, we expressed all our BioBricks. The FG repeats gene/MAP protein and the Star Magainin 1 were expressed in BL21(DE3) and Shuffle cells.  We had difficulty running a western blot on the crude whole cell lysate from the weekend, with nothing showing up during the ECL image development.
We will continue to troubleshoot this issue.

 

Week 14– 22 September

The Western Blot issue was somewhat resolved by using all of our parent lab’s reagents. It appears that our technique is correct, and there must have been an issue with our reagents or buffers. We suspect our PVDF membrane either has expired or is otherwise off. However, we will run duplicates of each gel from now on in order to determine the cause of our previous issues.
We continued the RGD cloning. We were having technical difficulties with the RGD to pSB1C3 operation. While the PCR product is visible on a gel, the gel extracted product is not. It is likely because the yield of the PCR is too low, which means that because of product loss during gel extraction, we don’t have enough DNA left to see on the subseqeuent gel.

 

Week 15 – 29 September

Successfully optimized the RGD to pSB1C3 PCR reaction by stepping through different annealing temperatures. By lowering the annealing temperature by 4 C, we were able to improve yield of the reaction.
We thought we might be able to perform the ligation, but when double digesting the linear magainin 1 gene in pSB1C3 with AgeI and PstI, three bands showed up on the blot. There should only be two. It is not clear why this is, and it may be due to contamination of the linear mag 1 plasmid. Although we could put more resources into this, we decided given the limited time, we would focus our efforts on other tasks, including protein expression and characterization and the remaining cloning task.
Having prepared all the vectors and inserts, we performed a ligation between RGD and pGEX-6P3 and RGD and pSB1C3.
The Western Blots from last week were inconclusive. There appeared to be his-tagged proteins in some of the cultures, but the staining was splotchy, not a clear band. Also, it was not clear which lanes had the staining.
On our advisors’ advice (H.C. Cheng), we performed a mini-scale his-tag purification on all cultures using Ni-NTA beads in Eppendorf tubes. We will run western blots on these purified samples next week.

 

Week 16 – 6 October

The results of the mini-purification were positive, showing his-tag staining in a western blot in the cultures. We decided to culture more bacteria as the mini purification did not give us enough sample last time for downstream analysis, as we had run out of cell pellet.
After the re-expression, we did a second mini-purification, this time using more (300 uL) of Ni-NTA beads.

 

Week 17 – 13 October

The second mini-purification purification appeared to work well on a his-tag Western Blot. We therefore set out to characterise our protein with mass spec, yielding the in-gel digestion results on the results page.