Team:EPFL/Notebook-2

iGEM EPFL 2018

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:

  1. 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
    4. 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

Image
Negative control (cells that were not transfected with the plasmid). As expected, no colonies have grown
Image
Here were plated the transfected cells. No culture have grown showing that the plasmids should have been degraded when staying too long at room temperature.

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 yieldTM 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)
Plasmid pMC_sfGFP-BsaI_noT7 puc19 CFP carrying plasmid mCherry carrying plasmid SYFP2 containing plasmid
Resistance Ampicilin Ampicilin Chloramphenicol Chloramphenicol Chloramphenicol
Vector - - pSB1C3 pSB1C3 pSB1C3

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 transf with Laurine + postivie control