Biomod/2013/Harvard/lab

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(Week 2)
(Week 2)
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The image below shows all of the GLucCaM and GLuc constructs that we have created by the end of Week 2. They were all inserted into the pDIMC8 backbone, which contains a chloramphenicol resistance gene, a multiple cloning site, and origin of replication, to create a plasmid that can be expressed by ''E. coli''.  
The image below shows all of the GLucCaM and GLuc constructs that we have created by the end of Week 2. They were all inserted into the pDIMC8 backbone, which contains a chloramphenicol resistance gene, a multiple cloning site, and origin of replication, to create a plasmid that can be expressed by ''E. coli''.  
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[[Image: GLucCam_Plasmids_Week2.png|thumb|left|300px|GLucCaM Plasmids]]
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[[Image: GLucCam_Plasmids_Week2.png|thumb|left|295px|GLucCaM Plasmids]]
[[Image: GLuc_Plasmids_Week2.png|thumb|right|290px|GLuc Plasmids]]
[[Image: GLuc_Plasmids_Week2.png|thumb|right|290px|GLuc Plasmids]]

Revision as of 16:29, 1 July 2013

Laboratory Notebook

Contents


Week 1

6/10/13

General BioMOD Cloning

purpose: clone for DH5α pDIMC8 BlaCaM His and DH5α pDIMC8 DsbA BlaCaM
  1. 5 mL LB
  2. 100µL 20% glucose
  3. Cm (34 mg/mL working stock), 7.35 µL of this, final concentration 50 µg/mL
  4. Add cells
  5. Inoculate overnight

6/11/13

I. Bacterial Solution: LB Agar Plates

purpose: to make agar plates for 1L LB-agar
  1. Mix stock
    1. 960mL DI water (860 mL if using glucose optional)
    2. 10g of Tryptone
    3. 10g of NaCl
    4. 5g yeast extract
    5. 15g agar
  2. Autoclave
    1. liquid (not dry) 30 min. sterilization/20 min drying
    2. cool to ~50°C in water bath
  3. Done under flame
    1. Add glucose - final concentration 20% glucose, if using 20% glucose then 100 mL, optional
    2. Add antibiotics to the specified final concentration. e.g. Cm: 50 μg/mL. Working stock: 34 mg/mL ⇒ 735 μL per 500 mL
    3. Pour onto the petri dishes, pre-mark them by type, e.g. : two black lines: Cm, red line: glucose
    4. Store

II. Experiment II from 6/10/13 continued: Miniprep (QIAGen)

purpose: to purify plasmids from our clone colonies
We followed MiniPrep
  1. transfer cell cultures into centrifuge tubes. Be careful not to drop the tip in.
  2. Centrifuge for 5 min. at 4000 rpm. N.B. make sure things are balanced.
  3. Dump the supernatant into old tube. Later add bleach before pouring down sink.
  1. Resuspend pellets and tansfer to 1.5mL labeled tube with 250μL P1 Buffer.
  2. 250 μL P2 Buffer (blue color): mix thoroughly.
  3. Time since step 2 must be less than 4 minutes: add 350μL of N3 Buffer. Mix thoroughly.
  4. Centrifuge at 13,000 rpm for 10 minutes.
  5. Apply the supernatant to QIAprep spin column.
  6. Centrifuge 1 minute. Discard flow-through.
  7. Wash w/ 750 μL Buffer PE.
  8. Centrifuge 1 minute. Discard flow-through.
  9. 1 min additional centrifuge.
  10. Column into 1.5 microcentrifuge tube.
  11. Elute with 50 μL water. 5 minute rest.
  12. Centrifuge 1 minute.

III. NanoDrop

We followed NanoDrop

IV. PCR Amplification: HercII PCR

Purpose: make backbone vectors for our Gibson reaction from pDIMC8 DsbA BlaCam. Will make:
  • pDIMC8 AIDA
  • pDIMC8 DsbA GLucCaM
  • pDIMC8 GLucCaM His
  • pDIMC8 DsbA GLucCaM His

Forward/Reverse Primer Chart:

Forward Reverse Key
AIDA pDIMC8 gib - for pDIMC8 gib - rev FW
DsbA GLucCam pDIMC8 gib - for pDIMC8 DsbA gib - rev CE
GLucCaM His pDIMC8 addHis - for pDIMC8 gib - rev TRN
DsbA GLucCaM His pDIMC8 addHis - for pDIMC8 DsbA gib - rev DZ

Image:Harvard BioDesign PCR 6-11-2013.png

We followed Herc II PCR protocol.

Week 2

Transformed and grew up colonies of cells with CE, TN, and DZ plasmids.

  • Realized that our TN plasmids were assembled with incorrect primers.
    • The resulting plasmids almost certainly do not have the genes we want inserted.
  • Used Qiagen MiniPrep kit to extract DNA from cells
    • Sent DNA off to have our plasmids sequenced to check for correctness.
    • Only send a few colonies of DZ and CE cells -- TN was not worth checking

Results:

  • The DZ plasmid was correctly assembled in one of our sequences
    • The other had a single mutation at residue 31 of GLucCaM gene
  • One of the CE sequences was confirmed as well
    • The other produced a sequence of too low quality -- likely not a correct plasmid

We started work on GLuc plasmids that contain only the G-Luciferase gene without the Calmodulin insertion. These will be used as controls during our future experiments.

  • Assembled plasmids with the same combination of His and DsbA tags as our GLucCaM plasmids.

The image below shows all of the GLucCaM and GLuc constructs that we have created by the end of Week 2. They were all inserted into the pDIMC8 backbone, which contains a chloramphenicol resistance gene, a multiple cloning site, and origin of replication, to create a plasmid that can be expressed by E. coli.

GLucCaM Plasmids
GLucCaM Plasmids
GLuc Plasmids
GLuc Plasmids

Sequencing results of the GLuc plasmids were good

  • Only the EF construction failed to return a correct sequence
    • Sent off 8 more colonies of EF for sequencing in the hope of finding a correct plasmid

The following table summarizes the progress we have made thus far with the GLucCam and GLuc plasmids.

Received AIDA autotransporter gene and began construction of plasmids containing AIDA and AIDA+insert (Bla, CaM, BlaCaM) fusions for bacterial display.

Week 3

To start off the week, we received our sequencing results for EF from Friday and found that the EF2-2, EF2-4, EF2-8, and EF2-6 samples had been successfully transformed with only noncoding mutations ( @902 A insert; @1017 G -> A mutation)

Continued construction of bacterial display plasmids.

  • Lots of assembly to be done here.
  • The gene that we want displayed must be inserted after the signal sequence and before linker region of the AIDA construct.
    • All of that must be put within our pDIMC8 vector.
  • This week was spent mainly trying to resolve cloning difficulties while creating our display plasmids.
    • Attempts at a 4 part assembly failed, and we also had to reconstruct a vector that refused to be modified.
  • We hope to have AIDA-Bla/CaM/BlaCaM fusions completed by the end of the short week next week.

We also began discussing how we will perform directed evolution once we have our display functioning.

  • Consensus seems to be on ordering an error-prone PCR kit because of its speed and ease of use

Week 4

Input: Our current focus is inserting Bla/CaM/BlaCaM into our backbone, which is the AIDA gene split and fused into the pDIMC8 vector

  • We are doing the assembly in two steps because Gibson assembling them all at once did not seem to work
    • This should be a more time-consuming, but more effective process (and hopefully successful)
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