Friday, 20/07/18

INSERT PCR WITH TAQ POLYMERASE

Aim: The Aim of this experience is to produce the hexahistidine insert with a PCR using TAQ.

Note:The primers we use from now on were diluted 10 times by mixing 10μlof the original primers with 90μl of water

1. PCR mix:

Green taq master mix (2X)	10μl
HexaHisForwardInsert	1μl
HexaHisReverseInsert	1μl
nuclease free water	8μl
total	20μl

2. PCR Cycle

Green taq master mix (2X)	10μl
HexaHisForwardInsert	1μl
HexaHisReverseInsert	1μl
nuclease free water	8μl
total	20μl

INSERT PCR WITH PHUSION POLYMERASE

Aim: The Aim of this experience is to produce the hexahistidine insert with a PCR using Phusion.

1. PCR mix in μl

Forward Primer	1μl
Reverse Primer	1μl
dNTPs (10mM)	0.4μl
Phusion Buffer	4μl
Phusion polymerase	0.2μl
H2O	13.4μl
Total	20μl

2. PCR cycle

STEP	Temp	Duration
Initial Denaturation	98	30s
Denaturation	98	10s	30 cycles
Annealing	72	30s
Extension	72	30s
Final Extension	72	1min
Hold	4

BACKBONE PCR

Aim: The Aim here is to amplify the backbone plasmid of encapsulin with a PCR.

1. PCR mix

Template 1ng/μl	1μl
Forward Primer 10mM	1μl
Reverse Primer 10mM	1μl
dNTPs (10mM)	0.4μl
Phusion Buffer	4μl
Phusion polymerase	0.2μl
H2O	12.6μl
Total	20μl

2. PCR cycle

STEP	Temp	Duration
Initial Denaturation	98	30s
Denaturation	98	8s	30 cycles
Annealing	63	25s
Extension	72	165s
Final Extension	72	10min
Hold	4-10

Results:

PCR gel electrophoresis of the insert PCR with Taq and Phusion

The PCR product were run on a 2% agarose gel, at 100V for 30 minutes.

The ladder is the o'generuler low range.

The first lane contains the PCR product of the insert with Taq, the 2nd lane contains the PCR product of the insert with Phusion. The 3rd lane is the forward insert primer and the last lane the reverse insert primer. 2μl of the Phusion PCR product and the primers were loaded with 2μl of loading dye. 4μl of the Taq PCR product, as well as the ladder, were loaded on the gel.

We can see a signal for the inserts that seem to be the right size as they are both next to the 100bp band of the ladder, the expected size for the insert is of 90 bp. It looks like Taq does work better than Phusion because the signal is stronger for Taq. It is also noticeable that the insert migrated less far than the primers which is what we expected, the primers were only 54 and 60 bp long.

PCR gel electrophoresis of the insert PCR with Taq and Phusion

Discussion

The PCR product for the backbone was run on a 1% agarose gel for 45 minutes at 100V.

2μl of PCR product were loaded with 2μ of thermo scientific 6X loading dye in the two lanes next to the generuler express DNA ladder. There are faint signals at around 100bp, the primers are supposed to be 28 and 30 bp long, So it's hard to tell what these can be. There is no trace of the backbone DNA, template, or PCR product.

Monday, 23/07/18

Since the last PCR didn't work we will try to change the temperature, by doing a gradient PCR with several temperature values

BackBone PCR

1. PCR mix

Template 1ng/μl	1μl
Forward Primer 10mM	1.25μl
Reverse Primer 10mM	1.25μl
dNTPs (10mM)	0.5μl
Q5 Buffer	5μl
Q5 polymerase	0.25μl
H2O	15.75μl
Total	25μl

2. PCR Cycle

STEP	Duration		Temp	Temp	Temp	Temp	Temp
Initial Denaturation	30s		98	98	98	98	98
Denaturation	8s	30 cycles	98	98	98	98	98
Annealing	25s		62	64	66	68	70
Extension	165s		72	72	72	72	72
Final Extension	2min		72	72	72	72	72
Hold			4	4	4	4	4

Results

The PCR product for the backbone was run on a 1% agarose gel for 45 minutes at 100V.

2.5µl of PCR product were loaded onto the gel with 2.5µl of thermo scientific 6X loading dye. 5µl of the o'gene ruler express ladder were loaded onto the gel.

As we can see there are no signal for the 5500 bp backbone we were expecting, which means that the PCR didn't work for any of the temperatures.

we can see signals below the 100bp mark of the ladder, these are probably the primers.

Tuesday, 24/07

We tried another PCR for the backbone but this time using a different template for the backbone. We had two different template.

• Template #1, comes from an aliquot of the backbone template at 844 ng/µl. It is labeled with encap sample 1 in the freezer.
• Template #2 comes from an aliquot of the backbone template at 350.8 ng/µl. It is labeled with encap sample 2 in the freezer.

Both samples were diluted to a final concentration of 1ng/µl

We recreated the following PCR mix, 3 times for each DNA template. for a total of 6 pcr mixes.

and ran the following gradient cycle for both sets of 3 PCR mix

Results:

We can see that the PCR worked at least once for each template. Surprisingly, the only time the PCR worked for template#1 was with an annealing temperature of 68°C while the calculated annealing temperature for the primers was 68°C. For template#2 the PCR worked for 66°C but we can also see a faint signal for the PCR with an annealing temperature of 68°C. However, it is clear that the PCR worked better with an annealing temperature of 66°C.

Conclusion

If you want to do a PCR amplification of the backbone, use an annealing temperature of 68°C if you use the template diluted from the 844ng/µl aliquot (encap sample1) and use an annealing temperature of 66°C if you want to use the template diluted from the 350.8ng/µl aliquot (encap sample2).

Notebook week 3 (23/07/18)

FRIDAY, 7/27/2018

Inoculating cultures, and miniprep

Aim: The aim of this experience is to grow liquid cell cultures from our transformed cells so we can use them to purify plasmid DNA later, and do glycerol stocks

Protocol: Inoculating cultures

We grew 2 different liquid cultures with 2 colonies from our transformed bacteria plate, as well as a negative control with only LB medium and ampicilin but no cells. They were incubated overnight at 37°C and shaked at 225 rpm.

Results:

SATURDAY, 7/28/2018

Isolation of plasmid DNA

Goals:

• To purify plasmid DNA from E.coli cells
• To quantify the amount of purified plasmid
• To prepare a glycerol stock of bacteria for long term storage

1.Plasmid Purification

Protocol: Plasmid miniprep

We performed 2 different MiniPrep reactions from our 2 different cell cultures, and obtained two different samples of purified plasmid

Results: Measurements of the purified plasmids with the nanodrop lite yielded the following results

Measurements were done with the nanodrop lite, using 1μl of nuclease-free water as blank, and 1μl of purified plasmid for the measurement.

Purified plasmids from cell culture #1:

• Concentration:14.6 ng/μl
• A260/280:1.72

Purified plamids from cell culture #2:

• Concentration:16.1 ng/μl
• A260/280:1.84

The concentration of purified plasmids are very low for both samples. Probably too low for being useful. We should perform another plasmid purification with more cell culture. Here we only purified 600μl from the cell culture. Next time we can try purifying a larger volume.

Notebook week 4 (30/07/18)

Monday, 30/07/18

Today we did another cell culture, like last time but with 4ml so we would take 500μl to make a glycerol stock of the cells and use the rest to purify the plasmids

Protocol: Inoculating cultures

• We did 2 liquid cell cultures with 2 different colonies from our plate. As well as a negative control

Tuesday, 31/07/18

1.Preparation of glycerol stocks

Aim:Make glycerol stocks of the cell cultures that grew overnight

Protocol:Glycerol stock preparation

We did a glycerol stock of both cell cultures in 2 different tubes, that were placed in the -80°C freezer

2.Isolation of plasmid DNA from a larger volume of cell culture

Aim:To try the purification of the plasmid DNA again, but with a larger volume of cell culture

Protocol Plamid miniprep

This time, we centrifuged 3ml of cell culture, discarded the supernatant and resuspended the cell pellet in 600ul of water to perform the miniprep

We finally purified 3ml from each cell cultures that were grown last night

Results: Measurements of the purified plasmids with the nanodrop lite yielded the following results

Measurements were done with the nanodrop lite, using 1μl of nuclease-free water as blank, and 1μl of purified plasmid for the measurement

Purified plasmids from cell culture #1:

• Concentration: 71,1 ng/μl
• A260/280: 1.72

Purified plasmids from cell culture #2

• Concentration: 167.5 ng/μl
• A260/280: 1.51

The concentrations are better than last time, However the A260/280 ratios are a bit low, especially for the second sample. This might lead to problems because of impurities in the samples.

Notebook week 5 (06/08/18)

MONDAY, 8/6/2018

Competent cell transformation: pMC_sfGFP-BsaI_noT7 plasmid into DH10β competent E.coli cells

pMC_sfGFP-BsaI_noT7 plasmid contains the sfGFP sequence which we will insert into our modified encapsulin plasmid in order to check take up by DCs The cell transfection will allow us to produce new plasmids, but first we have to check if the plasmid is still usable as it was stored quite long at room temperature.

We used the competent cell transformation protocol that we slightly adapted to our needs. Here are the modifications we made:

Amounts in µl	Transformation	Transformation control
Competent cells (in tube)	50	50
plasmids	5	-
Nuclease free water	-	1

2. Heat shock the cells at 42 °C for up to 45s (we let them 45s). Immediately transfer the tube back on ice for minimum 2 min (we made 3 min)
3. We did not add cell medium to the mixture because it is not needed when working with cells rendered resistant to Ampicilin
Ampicilin directly kills cells which are not resistant to it --> not the case of other antibiotics the outgrowth step serves to prevent growth of other types of bacteria that could then destroy the ampicilin on the plate and then there is a risk that not resistant bacteria grow
4. We spread 50 uL each time on LB+ Amp plates using spreader we made ourselves and sterilised

TUESDAY, 8/7/2018

Results of the transformation of DH10β competent E.coli cells with pMC_sfGFP-BsaI_noT7 plasmid

Inoculation of cultures from glycerol stocks of cells (DH5α or DH10β) containing HexaHistidine-encap plasmids

Cultures were inoculated following this protocol

We had glycerol stock from 2 different colonies so we made 2 different tubes as well as a negative control in which we just put LB medium and Ampicilin.

We used 1000x Ampicilin and as we need 1000x less Ampicilin than medium we put 3uL of Ampicilin in each tube.

Purification of the plasmids of the cultures from glycerol stocks of cells (DH5α or DH10β) containing HexaHistidine-encap plasmids

The plasmids were purified using the Pure yield^TM plasmid miniprep kit from promega following the corresponding protocol.

The resulting of the purification from the two different colonies are as follows:

• colony 1:

260/280: 1,75
concentration:50,4 ng/uL

• colony 2:

260/2801,79
concentration:41,2 ng/uL

Test of the efficiency of DH-10 β competent E.coli cells

puC19 Plasmid, pMC65, pMC116 were heat shocked into three different competent cell tubes and plated on Amp plates. The plates were incubated overnight at 37 degrees

THURSDAY, 8/9/2018

Result of the test of the efficiency of DH-10 β competent E.coli cells

No colonies were found on the plates, it may be that the cells used are not competent.

Competent cell transformation using DH5α competent E.coli cells

The competent cells were given to us by the LBNC lab and their efficiency was already assessed.

We transformed the cells with the 3 following plasmids:

• pMC_sfGFP-BsaI_noT7

(contains sfGFP) --> we called it p65

• pSIREN_U6-Cpf1sgRNA

(contains RFP) --> we called it p116

• puc19 (a standard control plasmid)

We followed this protocol (https://benchling.com/lbnc/f/OOhQKnto-encapsulin/prt-b5aeAD4Y-competent-cell- transformation/edit) to transfrom the cells and added a transformation control (50uL of competent cells with 5uL of nuclease free water).

FRIDAY, 8/10/2018

Results of the cell transformation using DH5α competent E.coli cells

No colonies were found on any of the plates. As the cells have already shown efficiency in other experiments, this could mean that the plasmids that stayed for weeks are room temperature (p116 and p65) were all degraded and that the puc19 whose expiration date is 2017 is not viable anymore.

Competent cell transformation using DH5α competent E.coli cells

The competent cells were given to us by the LBNC and their efficiency was already assessed.

We transformed the cells with the 3 following plasmids:

• pMC_sfGFP-BsaI_noT7

(contains sfGFP) --> we called it p65
(this one was given to us by the LBNC and was stored in good conditions)

• plasmid carring mCherry from iGEM distribution kit
• plasmid carring Cyan Fluorescent Protein from iGEM distribution kit
• plasmid carring YGFP from iGEM distribution kit

We followed this protocol (https://benchling.com/lbnc/f/OOhQKnto-encapsulin/prt-b5aeAD4Y-competent-cell- transformation/edit) but as we were not well organized we were not able to start directly when we got the cells from the LBNC and we let them thaw a bit on ice and then refroze them in the -20 °C freezer before finally thaw them on ice and use them. Moreover we were using 1 plasmid conferring Ampicilin resistance and 2 conferring chlorampenicol resistance but we did the outgrowth step for the 3, although we should not have done it for the 1 conferring ampicilin resistance. Finally, the quantity of DNA we used to transform the cells with the plasmids from iGEM's distribution kit was very low; indeed iGEM suggests to dilute the dried DNA from the distribution kit in 10uL water and then do the transformation with 1uL which corresponds to 200-300pg of DNA.

SATURDAY, 8/11/2018

Result of the competent cell transformation using DH5α competent E.coli cells

No colonies were found.

Notebook week 6 (13/08/18)

Cells we used:DH5α competent E.coli cells (some from Ivan and some from Michael)

Plasmids we used:

• pMC_sfGFP-BsaI_noT7 (https://benchling.com/lbnc/f/2wg8QdVJ-introduction/seq-Uj4WhTuX-pmc_sfgfp-bsai_not7/edit)
• puc19
• CFP carrying plasmid (http://parts.igem.org/Part:BBa_K404319)
• mCherry carrying plasmid (http://parts.igem.org/Part:BBa_J06504)
• SYFP2 containing plasmid (http://parts.igem.org/Part:BBa_K864100)

We followed iGEM's single tube transformation protocol (http://parts.igem.org/Help:Protocols/Transformation) but we put only 30uL of cells in each tube and used LB medium instead of SOC.

We did the mistake to also put medium to the ampicilin resistant bacteria, they were put in the incubator for ~1min before she realized it. Then we took them, put them quickly on ice (which was a bad idea), put them in the microcentrifuge and centrifuge them for 3min at 6,8g. We removed the supernatant and vortex them a bit and then plate the resulting 160uL.

Result of the competent cell transformation

We had colonies only on the plate where we put the cells transfected with pMC_sfGFP-BsaI_noT7 (https://benchling.com/lbnc/f/2wg8QdVJ-introduction/seq-Uj4WhTuX-pmc_sfgfp-bsai_not7/edit).

Pouring LB Agar plates

For 200mL of LB Agar mixture (~10 plates) --> 3g Agar, 5g of LB
! to cover the erlenmeyer after the autoclave!

Competent cell transfection with Laurine + postivie control

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