Split IFP & GFP  - Week 1

1        CPXR extraction

Extraction of CPXR from genome of E. Coli strain K-12 MG1655

1.1       Material and methods

NEB Phusion HF PCR Protocol (see protocol), with corresponding primers (cf primers datasheet) for amplification. 

1.2       Results

1.2.1     Data

1.2.2     Interpretation

CPXR expected size : 699 bp

Amplification of CPXR worked as expected.

2        PCR for amplification of iGEM plasmid pSB1C3 and for addition of overlapps to CPXR

Amplification of pSB1C1 backbone from Cambridge biobrick K325219. This backbone contains the RBS, an arabinose-inducible pBAD promoter, iGEM prefix (containing ECORI and xbaI restriction sites) and suffix (contaning SpeI and PstI restriction sites), a chloramphenicol resistance gene, and an origin of replicaiton. Additon of overlapp to CPXR in order to make a Gisbon assembly of the backbone and the CPXR

2.1       Material and methods

NEB Phusion PCR and suitable primers.

2.2       Results

2.2.1     Data

2.2.2     Interpretation

We obtained the expected amplicon, and are now able to make a Gibson assembly to fuse the backbone amplicon and the CPXR.

1      Gibson assembly and colony PCR of pCPXR

Fusion of pSB1C3 backbone and CPXR

1.1       Material and methods

Gibson assembly kit (see protocol) for the Gibson assembly, and Taq polymerase for colony PCR (see protocol)

1.2       Results

1.2.1     Data

Colonies were obtained and analyzed by colony PCR, using VF2 and VR primers from iGEM

1.2.2     Interpretation

The amplicons have the expected size, meaning the Gibson assmbly worked and that we very likely obtained pCPXR (plasmid with pSB1C3 backbone and CPXR). Sequencing will reveal if the plasmid contain any mutation.

2        Sequencing of pCPXR

Confirmation of pCPXR sequence with Microsynth sequencing services.

2.1       Material and methods

We sent the plasmid with a concentration of 80 ng/μl and with premixed primers VR and VF2

2.2       Results

2.2.1     Data

We obtained the right sequence, excepted one deletion of a cytosine in the ara promoter at the position -41. This deletion appeared to be originally in the backbone pSB1C1 that we amplified from Cambridge biobrick K325219, without consequence on the transcription of the coding sequence.

3     Amplification of IFP1, IFP2 and pCPXR with suitable overlaps to fuse the split at the C or N terminal of CPXR

PCR amplification of fragments to fuse IFP1 and IFP2 at the C or N terminal of CPXR in pCPXR plasmid

3.1       Material and methods

NEB Phusion kit (see protocol) and suitable primers (see list). Template come from Michnick lab IFP PCA.

3.2       Results

3.2.1     Data

3.2.2     Interpretation

IFP1 C didn't work but the other ones have the expected size. We are going to try again IFP1 C in a gradient PCR

4        PCR gradient to obtain IFP1 C fragment

Amplification of IFP1 with overlap in order to insert it in pCPXR at the C terminal of CPXR

4.1       Material and methods

Gradient temperature was performed as the PCR didn’t work the first time. NEB Phusion kit, same procedure as last time

4.2       Results

4.2.1     Data

4.2.2     Interpretation

We finally obtain the amplicon, for each temperature.

Experiment 1: Transformation

Transform competent cells with plasmids recovered from Michnick’s and Waldor’s labs

1.1 Material and methods

14/07/14

Preparation of SOC: 3 x (20µl of glucose 20mM in 1ml SOB)

Transformation Protocol from iGEM HQ with 1 uL DNA in each Ependorf

Incubation times: 2h shaking at 37°C / 180 rpm and 2h resting on ice.

Plates set for incubation overnight at 37°C

15/07/14

Inoculated cultures in LB Chloramphenicol or Ampicillin (3ml)

Tubes left in the fridge all day

Incubation overnight at 37°C / 180 rpm

16/07/14

OD measurement

1.2 Results

1.2.1 Data

OD: 1. pYZNC A: 2.046

2. pYZNC B: 2.033

3. prLucNrLucC A: failed (overcrowded plate)

4. prLucNrLucC B: 2.072

5. prLuc A: 2.028

6. prLuc B: 2.055

7. pIFP 1 A: 1.950

8. pIFP 1 B: 1.953

9. pIFP 2 A: 1.954

10. pIFP 2 B :1.948

1.2.2 Interpretation

Tube 3 discarded, others used for miniprep and glycerol stock preparation

Experiment 2: Bioluminescence assay

Make a bioluminescence assay with the previously prepared cultures

2.1Material and methods

17.07.14:

Overnight cultures (3ml LB + chloramphenicol) inoculated with cells from a frozen glycerol stock from previously transformed competent cells (pYNZC A & B, prLucNrLucC B, prLuc A & B) were incubated at 37°C with shaking at 180 rpm). OD was measured the following day.

18.07.14:

The cultures were diluted 1:100 in 5mL fresh LB-Chloramphenicol containing 670ul of 100mM L-arabinose and grown for 3h on a rotary shaker to an OD 0.8-0.9. The cultures (500ul of each) were washed twice with phosphate buffered saline (PBS; pH 7), adjusted to an OD 0.4-0.5 (final volume of culture should be 1ml) and aliquoted into a white 96-well plates in triplicates.

Aliquots of native coelenterazine (clz) dissolved in ethanol (10mM) were stored at 80°C. Solution of glucose (30mM in bi-distilled water) were prepared and used at a concentration of 1mM. Prior to the assay, an aliquot of clz was thawed, diluted in PBS to a final concentration of 250uM, and incubated in the dark at room temperature (RT) for 1h. Clz was added to a final concentration of 7.5uM to the PBS-washed cells, which were subsequently incubated in the dark for 30 minutes at RT. The total luminescence of each well was then measured every two minute for a total of 10 min with an integration time of 1 sec using (WHICH MACHINE??). Immediatly thereafter, 10 ul of glucose solution was manually injected into each well and a second luminescence read was initiated as before.

2.2Results

2.2.1Data

2.2.2Interpretation

The measurements were taken too late. The substrate (clz) already started to be depleted. The positive control gave a very strong signal at the beginning, while the negative control gave a small or no signal. The construct itself gave a signal higher than the negative control but much lower than the negative control, meaning that the construct works but isn't optimal. The effect of the addition of glucose after 10 minutes can't be determined precisely. As there was no more clz in the wells after 6-7 minutes, the shut down of the luminescence observed in the construct wasn't necessarily due to glucose addition, as exprected. We cannot conclude on this experiment. We should start again and take measurements faster.

07.07.14-13.07.14

Competent Cells

Protocol : Thermocompetent cells protocol

First try – Not much care, all resulting tubes were thrown away

Second try – Lesser volume, more care to cold, poured liquid nitrogen right into box for freezing.

Results : Tests seem to indicate that our method for transformation was simply not adapted to our competent cell protocol. We do not have the appropriate sterile cold room to produce high efficiency competent cells. Therefore, for biobricks and gibson assemblies, we need to use commercial competent cells. For transformations with plasmid constructs where the concentration is in the scale of 1 ng/ul or more our competent cells are usable.

1 Experiment 1: Bioluminescence assay

Make a bioluminescence assay with the cultures transformed with plasmids from Waldor's lab

1.1 Material and methods

22.07.14: Overnight cultures (3ml LB + chloramphenicol) inoculated with cells from a frozen glycerol stock from previously transformed competent cells (pYNZC, prLucNrLucC, prLuc) were incubated at 37°C with shaking at 180 rpm). OD was measured the following day.

23.07.14: The cultures were diluted 1:100 in 5mL fresh LB-Chloramphenicol containing 670ul of 100mM L-arabinose and grown for 3h on a rotary shaker to an OD 0.6-0.7. The cultures (1ml of each) were washed twice with phosphate buffered saline (PBS; pH 7), (final volume of culture should be 1ml) and aliquoted into a white 96-well plates in triplicates.

Aliquots of native coelenterazine (clz) dissolved in ethanol (10mM) were stored at 80°C. Prior to the assay, an aliquot of clz was thawed, diluted in PBS to a final concentration of 250uM, and incubated in the dark at room temperature (RT) for 1h. Clz was added to the PBS-washed cells, at different concentrations: 7.5uM and 75uM for pYNZC and rLucNrLucC, 7.5uM, 15uM, 75uM and 150uM for rLuc, for a total volume of 300ul in each well (see table below). Cells were subsequently incubated in the dark for 5 minutes at RT. The total luminescence of each well was then measured every three minutes for a total of 30 min with an integration time of 1 sec.

1.2Results

1.2.1Data

OD from overnight cultures :

pYNZC A: 1.916

pYNZC B: 1.889

rLucNrLucC: 1.794

rLuc A: 1.878

rLucB: 1.701

OD after 3h shaking (after addition of arabinose):

pYNZC A: 0.683

pYNZC B: 0.634

rLucNrLucC: 0.608

rLuc A: 0.600

rLucB: 0.550

[GRAPHS]

1.2.2Interpretation

After addition of coelenterazine to the cells in the plate, wells were full and the cultures may have spread between the wells. Our results aren't as expected, as the signal in the negative control is higher than in the pYNZC construct.

The measurements should be taken in an opaque 96-well plate, from the bottom of the plate. The wells shouldn't be filled completely to avoid spreading.

The concentration of bacteria should be taken in account to make the gradient of concentrations.

2 Experiment 2: PCR extraction of fragments of interest

Extraction of sequences of interest from plasmids to proceed to Gibson assembly of the final construct: CheYrLucNCheZrLucC.

2.1 Material and methods

PCR kit (NEB), see protocol from kit

Minipreps from following plasmids: CheY (Bba_K569017), CheZ (Bba_K629003), rLucN (YNZC, Waldor), rLucC (YNZC, Waldor)

Primers (TABLE)

Preparation of the tubes (in duplicates) for 50uL reaction:

*MasterMix: for one tube: 9uL HF buffer, 0.9uL dNTP, 0.5uL polymerase

Control tubes:

*Control MasterMix: for one tube: 9uL HF buffer, 0.9uL dNTP

PCR program according to Phusion Polymerase protocol

2.2 Material and methods

1.2.1 Data

1  Gibson assembly and colony PCR for IFP2-pCPXR (at the C and N terminal)

Assembly of IFP2 at the C ter or N ter of CPXR in pCPXR by Gibson assembly. Colony PCR is useful to check if competent cells were transformed by our expected Gibson Assembly product and not by the empty plasmid pCPXR.

We did the GA with PCR product of pCPXR opened with corresponding primers and amplified insert, but we didn’t get rid of the pCPXR DNA template so competent cells may have been transformed with empty vectors thus acquiring chloramphenicol resistance. This is why we obtained so many negative colonies. DPN1 digestion of our PCR product would have removed the plasmids template.

1.1       Material and methods

We used VR ans VF2 sequencing primers.

1. pick a colony and pouf pouf in 10ul tubes of water. Directly put the tip in 3ml of LB with chloramphenicol in culture tubes and put at 37°.
2. Boil the 10ul tubes of water containing the picked bacteria at 95° for 10 minutes. It will be your « DNA template ».
3. Prepare the PCR mix for Taq polymerase, with 1ul of DNA template (NEB Taq protocol)

1.2       Results

1.2.1     Data

We also loaded PCR product of pCPXR "opened" as negative control, in order to compare the size of our GA product and pCPXR and see if it contains the IFP2. Colonies that are thought to contain the insert IFP2 are:

• IFP2 C terminal: 1, 4, 5, 6, 7, 9, 10, 11, 12
• IFP2 N terminal: 1, 2, 3, 4, 6, 9, 12

1.2.2     Interpretation

By observing the difference of size between the negative control (PCR of pCPXR that doesn’t contain the insert) and the colony PCR fragment, we see that we obtained some colonies containing empty vectors and some that contains very likely IFP2 insert. Further anlyisis and sequencing will confirm it.

2        PCR amplification of IFP2 in the plasmid thought to contain IFP2

Amplification of IFP2 from IFP2-pCPXR plasmid, to confirm that our GA product contains the insert IFP2.

2.1       Material and methods

We used primers previously used to amplify and to add an overlap to IFP2 Cter and Nter respectively. As the « tail » of the primers were now annealing the plasmid, one could think that changing the Annealing temp was necessary but you can keep the same that you used the first time. Unfortunately some of the PCR tubes opend in the PCR machine so we couldn’t analyzed all of the plasmid.

2.2       Results

2.2.1     Data

2.2.2     Interpretation

All of the plasmids analysed contain the insert IFP2 and the size is correct (600 bp). Our GA worked, at least for the colonies that were analyzed on this gel. We are now ready for sequencing

3        Sequencing IFP2-pCPXR plasmid

To check if our GA products, pCPXR fused to IFP2 at its Cter or Nter don’t contain any mutation. We sent sample 9 for IFP2 Nter and sample 10 for C terminal.

3.1       Material and methods

Microsynth Barcode and a plasmid concentartion of 80 ng/ul

3.2       Results

3.2.1     Data

No mutation for IFP2 at the C terminal. One mutation in the RBS of IFP2 at the Nterminal, position -18 a A instead of a T. It shouldn't matter. 

4        Colony PCR for IFP1-pCPXR (at the C and N terminal)

Assembly of IFP1 at the C ter or N ter of CPXR in pCPXR by Gibson assembly. Colony PCR is useful to check if competent cells were transformed by our expected Gibson Assembly product and not by the empty plasmid pCPXR.

4.1       Material and methods

(See above for protocol)

WARNING : We should always do a negative control for the colony PCR with Bacteria transformed with the empty vectors, not a PCR of an empty plasmid. Otherwise you cannot make any conclusion if the colony PCR fails or if the Gibson Assembly fails. You can compare the colony PCR quality of an expected product (pCPXR) and colony PCR of your new Gibson Assembly.

4.2       Results

4.2.1     Data

In blue: IFP1 at the C terminal. In red: IFP2 at the N terminal

4.2.2     Interpretation

Colonies that are thought to contain IFP1:

• IFP1 at the C terminal: all
• IFP1 at the N terminal: 3, 4, 6, 7, 8, 9, 10

5        PCR amplification of IFP1 in IFP1-pCPXR plasmid (after mini-prep)

Colony PCR was not really conculsive so we decided to min-prep all the colonies and analyse the obtained plasmids.

5.1       Material and methods

IFP1 amplification of the obtained plasmid was done with primers previously used to add to IFP1 the overlapp (IFP1_Cter and IFP1_Nter). We used Taq polymerase.

5.2       Results

5.2.1     Data

IFP1 amplification from colonies supposed to have the plasmid pCPXR with IFP1 at its Nterminal.

Amplicon expected size is 463 bp, which correspond to our results. Colonie 5 apparently doesn’t have IFP1. “+” means that we amplified IFP1 from the plasmid known to contain this sequence. We also obtained a band at 850 bp for all the colonies that have the IFP1 fragment. Interestingly we also obtained this band in the positive control…

IFP1 amplification from colonies supposed to have the plasmid pCPXR with IFP1 at its Cterminal.

Expected size: 484 bp which correspond to our results.

5.2.2     Interpretation

We very likely contain pIFP1-CPXR and pCPXR-IFP1 and further sequencing will confirm it.

1        Restriction Analysis of IFP1 Cter and Nter

To check if the plasmid contains IFP1 in our Gibson Assembly product, by digesting the plasmid with NheI and SpeI and see if we obtain the expected sizes.

1.1       Material and methods

• 500 ng of DNA (plasmid)
• 0.5 ul of SpeI and 0.5 ul NheI (should be added at the end)
• 5 ul of cut smart buffer
• Add water to 50 ul

Incubate for one hour at 37°

Load on gel

1.2       Results

1.2.1     Data

Plasmid that have been digested in 2 fragments of size 1169 bp and 2773 bp contains a fragment, which is likely IFP1 (expected size). First gel reveals that all the plasmid supposed to contain IFP1 at the Cterminal of CPXR likely have IFP1. The second gel reveals that all colonies exept the 1^st one likely contains IFP1 at the Nterminal. Further sequencing will confirm it.

2        IFP1 Cter and Nter sequencing

To check if the inserts IFP1 Cter and Nter, IFP2 Cter and Nter are present in the Gibson Assembly products (pCPXR fused by the insert below)

2.1       Material and methods

We will use the primer pBad For provided by mycrosynth (standard primer list) which will allow the sequencing from the end of the arabinose promoter. We will use also the VR primer to sequence Reverse from the suffix

• IFP1 Cter : 10 (90 ng/ul)
• IFP1 Nter : 9 (94 ng/ul)

2.2       Results

2.2.1     Data

•  9   IFP1 Nter : No deletion 
• 10   IFP1 Cter : one deletion in the linker, doesn’t change the reading frame of IFP1

Expected sequence                                      Our sequence

• GGG TCC TCC                                     GGA ---- TCC

  Glycine Ser Ser                                   Gylcine----Serine

2.2.2     Interpretation

3        Experiment 3: Title

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Name of the experimenter

Purpose of the experiment

3.1       Material and methods

3.2       Results

3.2.1     Data

Conclusion

1        PCR of pCPXR-IFP1 and pCPXR-IFP2 to put both fragment in the same vector

To do a new plasmid containing our two fragments of interest, CPXR-IFP1 and CPXR-IFP2 into each configuration (IFP1 at the C or N terminal of CPXR and IFP2 at the C or Nterminal of CPXR) to avoid cotransformation during further experiment.

1.1       Material and methods

Phusion PCR with a temperature gradient as our two first trial failed completely. Gradient temperature was achieved from 60° to 70°, the theorical temperature being arount 70°. pCPXR-IFP2 will be entirely amplified and IFP1-CPXR fragment will be amplified by PCR with suitable overlapping ends for further Gibson assembly reaction.

1.2       Results

1.2.1     Data

Every template has been amplified 7 times, each with a different annealing temperature, respectively from the first amplicons : 70°, 69.5°, 68.4°, 66.4°, 64°, 62°, 60°

1.2.2     Interpretation

Not all the annealing temperature worked but we obtained one amplicon of each IFP1-CPXR, CPXR-IFP1, pCPXR-IFP2 opened, pIFP2-CPXR opened.

2        Gibson assembly to make 4 different constructs containing both IFP1-CPXR and IFP2-CPXR in different configuration and colony PCR

Fusion of the insert IFP1-CPXR (IFP1 at the C or N terminal of CPXR) in the plasmid containing IFP2-CPXR (IFP2 at the C or N terminal of CPXR), to have our four constructs:

GA1: IFP1 at the Cterminal of CPXR and IFP2 at the Cterminal of CPXR

GA2: IFP1 at the Cterminal of CPXR and IFP2 at the Nterminal of CPXR

GA3: IFP1 at the Nterminal of CPXR and IFP2 at the Cterminal of CPXR

GA4: IFP1 at the Nterminal of CPXR and IFP2 at the Nterminal of CPXR

2.1       Material and methods

NEB Gibson assembly kit and NEB Taq polymerase for the colony PCR with primers VR and VF2

2.2       Results

We didn’t obtain any colonies for GA1 and GA3, and only a few for GA2 and GA4. We picked 13 colonies of GA4 and 10 colonies for GA.

2.2.1     Data

• Colony PCR of GA4 with VF2 and VR primers. Expectation is about 4050 with both IFP1-CPXR and IFP2-CPXR, and of about 3000 without one of them.

• Colony PCR of GA2 with VF2 and VR primers. Expectations are the same.

2.2.2     Interpretation

We can’t conclude anything with this ladder, and the difference between our wanted product and one that doesn’t contain both fragment of interest is of 1000 bases so we can’t reaaly see it on the gel. Mini-prep has been done and restriciton analysis will allow us to conclude if the colonies contained the expected product.

3        Restriction enzyme analysis of GA2 and GA4

Digestion of our constructs GA2 (containing IFP1 at the Cter of CPXR and IFP2 at the Nter) and GA4 (containing IFP1 at the Nter and IFP2 at the Nter) with enzymes SpeI and NHeI to see if they contain the expected fragments.

3.1       Material and methods

NEB protocol for restriction enzyme digestion was applied and enzyme SpeI and NheI were used.

3.2       Results

3.2.1     Data

• Restriction analysis of

Expected with the insert: 3273 bp + 2539 bp

With only CPXR-IFP1: 1200 bp + 3273 bp

With only CPXR-IFP2: 1400 bp + 3273 bp

• Restriction analysis of

Conclusion

Experiment 1: Arabinose promoter characterization

1.1       Material and methods

Cells with sfGFP CpxR & CpxR sfGFP constructs were cultured in different concentrations of arabinose to check the functionality of the promoter. The concentration of arabinose per sample is indicated in Table 1.

Table 1 - This table shows the concentration of Arabinose in each sample, as well as the quantity of medium and of Arabinose needed to reach it.

The negative control was the medium without cells, and the positive control was a sample of cells expressing sfGFP contitutively.

Procedure

Cells from glycerol stocks were grown in 3 mL LB selective medium (chloramphenicol) during 3h (37 ºC & shaking).

50 uL of cells were resuspended in a total volume of 5 mL of LB-chloramphenicol and Arabinose and incubated 5h at 37 ºC with shaking until an OD of ~1 was reached.
The samples were then washed twice with PBS to a final OD of 0.4 - 0.5.

Table 2 shows the quantity of PBS added to each sample (depending on the OD), as well as the OD after both PBS washes. This information is used to normalize the plate reader results.

 Table 2 - OD measurments of the samples can be seen, as well as the OD after the PBS washes.

Results

1.4.1     Data

https://static.igem.org/mediawiki/2014/0/06/Sfgfpcpxr_gradient.jpg

Figure 1 - Photo of the pelleted samples between PBS wash steps 1 and 2.

As can be seen in the image, there is a gradient of fluorescence intensity.

The results of the plate reader are depicted in Figure 2.

https://static.igem.org/mediawiki/2014/f/f5/Twotermscpxrgfp.jpg

Figure 2 - Graphs of the intensity curves of the samples. Cterm corresponds to CpxR sfGFP (sfGFP attached to CpxR's C temrinus) and Nterm corresponds to sfGFP CpxR.

1.4.2     Interpretation

As can be seen in the graph, the the results of the sfGFP CpxR sample was much better (closer to the expected results) than the one on the CpxR sfGFP, although in general, the curves grow.

It could be seen that adding too much arabinose decreases viability, so we considered that a concentration of 5 mM was the optimum arabinose concentration.