• Shelf organization: Acids, Proteins, Buffers, Gels, Agarose, Salts, Carbs
• Competent Cell Formation (K12)
  • See comp. cell prep protocol in VA iGEM 2016 Lab Protocols

Agenda:

• PrG Determination (1-4)
• Orthogonality Research
• Competent Cell Formation Cont. (K12)
• Growing K12 cells and make buffer (sterilized transformation buffer)
  • Plans to grow another batch with XL1 Blue tomorrow

Modeling:

• Research docking software and test

Competent Cells:

• Making Sterilized Transformation Buffer (500ml)
• Took out cells at 9:30am
• Transferred to 20°C at 11:00 PM, pour one of XL1-Blue and K12

Competent cells:

• Cultures left 18°C
• 11pm tonight: into LB + back into 18°C
• Sterilized filtration buffer

Protecting group:

• 2?:Phenyl acetyl protecting group, cleaved by penicillin g acylase
• Truncated from 5 PGs to 3
• 1: Dipeptide (maybe leu-leu), with a D aa on N-terminus
• A protease w/ cleavage activity acting on D aa is being researched for increased specificity

Modeling:

• AD4 returned zero errors w/ synthetase and leucine as ligand
• Still unsuccessful AG4 and AD4 returned
• Researching alternatives to direct another approach
• Unpacked Rosetta ligand modeling
• IGEM dock successful modeling; concerns about synthetase model

CRISPR

• Found biobrick to insert plasmid
• An alternative approach to silencing RNAs and transformation

Funding

• Anders finished half application for grant

Removing competent cells from 18°C
After 17 hrs:

• XL1 blue at an OD of 0.012
• K12 at an OD of 0.16

Put XL1 blue back into 18°C incubator, put K12 into 4°C fridge Competent cells:

• Removed XL1 blue at a OD of 0.13, centrifuged with buffer, added DMSO, froze with liquid nitrogen

Lab work:

• Made CAM plates
• Made SOC media
• Competency test

Decided on 4 different PrGs:

• Cbz leu, pro leu, phenyl acetyl leu, N-methoxy leu

Modeling:

• Corrected enzyme mistake, now using correct configuration of enzyme

Lab work:

• Competency test control plated at 4:50pm

Lab work:

• No growth on control plates

Lab work:

• JW cells subcultured on LB agar plate in 37°C incubator

Lab work:

• JW cells grown in overnight liquid culture at 37°C shaking

Lab work:

• JW cells frozen in glycerol stock at -80°C (5 tubes)

Lab work:

• Preculture of JW in 20 ml LB
• Made 50 ml of sterile LB and 0.1g of pro-leu

Lab work:

• Began pr-leu uptake test
• Take OD: 0.07 for each preculture, incubate
• Test for lawn growth or individual colonies on a spread plate (for CRISPR)

Lab work:

• Uptake test OD measurements
  • AM measurements: 1.137 for LB, 1.422 for LB + pro-leu
  • 7 pm measurements:1.104 for LB, 1.994 for LB + pro-leu
• CRISPR plate - lawn produced

Pr-leu uptake test:

• Test failed, bacteria were fixed by ethanol overnight and could not be lysed
• Restarted test, XL1-blue innoculated in LB at 5:20pm

Policies and Practices:

• Meeting with Rivanna river waste treatment plant
• Meeting with open bio labs

Tested spectrophotometer in the PLSB lab - Not consistent with results in Prof. Kozminski's lab Pr-leu uptake test:

• Failed again, cells did not lyse
• Started a new pr-leu uptake procedure

Wiki team meeting - wiki design Created standards for LC-MS Lab work:

• Transformed XL1-blue with Bba-K1218011 CRISPR Cas9 plasmid
• Transformed XL1-blue with BBa-B0010 forward terminator plasmid
• Transformed XL1-blue with RFP control plasmid

Lab work:

• Growth on CRISPR, terminator, and RFP plates, no growth on controls
• Cultures prepared for glycerol storage of transformed bacteria

Lab work:

• Optical densities taken for second round of pr-leu uptake and enzyme tests

Lab work:

• Optical densities taken again for second round of pr-leu uptake and enzyme tests

Lab work:

• Sample taken from enzyme reaction and frozen in -20° freezer

Lab work:

• Second sample taken from enzyme reaction and frozen in -20° freezer

Lab work:

• Redoing growth uptake test with aerated growth conditions

Lab work:

• OD measurements taken for growth test
• Extracted and purified E. coli genomic DNA via a minikit
• Began PCR on genomic DNA to obtain the leuS gene DNA

Lab work:

• Purified the PCR product
• Ran a gel to confirm product - bands ran together
• Took OD measurements for growth test

Lab work:

• Extractions performed for enzyme test
• Redo growth uptake test again

Growth Uptake Test Results
Supplement	Growth at 10:05am	Growth at 4:00pm	Growth at 9:08pm
1 None	None	None	None
1 None	None	None	None
3 None	None	None	None
1 Leu	None	Yes	Yes
2 Leu	None	Yes	Yes
3 Leu	None	Yes	Yes
1 Z 3	None	None	None
2 Z 3	None	None	None
3 Z 3	None	None	None
1 Z 8	None	None	None
2 Z 8	None	None	None
3 Z 8	None	None	None

Lab work:

• PCR performed on LeuS gene and product was purified

Lab work:

• Confirmatory digest performed on LeuS PCR product
• XL1-blue cells transformed with LeuS-ampR plasmid - plated and incubated at 10:45pm

Lab work:

• Inoculated precultures of LeuS and terminator transformed cells for miniprep and sequencing

Lab work:

• Redoing enzyme test (again) with a new protocol
• Performed a miniprep to obtain LeuS and terminator plasmids

Lab work:

• Sent off minipreped LeuS and terminator plasmid DNA for sequencing
• Performed confirmatory digests on LeuS and terminator plasmid DNA

Lab work:

• Performed PCR and PCR purification

Lab work:

• Talked with Professor Kozminski
• Set up another PCR reaction and PCR purification

Lab work:

• No colonies grew on the terminator transformation plate
• Restarted enzyme efficacy test

Lab work:

• 9:50am: Removed and stored a sample of the enzyme test
• Observed growth on the RFP, T1, and T2 plates, no growth on control
• Started precultures with colonies from the terminator 1 and 2 plates

Lab work:

• Performed gel electrophoresis, obtained wrong product
• Extracted DNA from gel
• Performed minipreps

Lab work:

• Confirmatory digest of minipreps from 7/15
• Performed a ligation reaction for T1 and T2

Lab work:

• Transformed XL1-blue cells with T1, T2, and an RFP control
• Extracted digested products from agarose gel
• Plated again using transformed cells from 12am at 1:30pm
• Transforme and plated new cells at 3:30pm
• Ligation reaction performed for T1 and T2 using DNA extracted from gel

Lab work:

• Transformed XL1-blue cells with the 2 ligation products (T1 with PCR, T2 with PCR)
• Began a preculture of XL1-blue cas9 cells in LB+cam at 5:45pm

Lab work:

• Performed a restriction digest
• Gel electrophoresis at 1:45am
• Gel weights: T2-173g, T1-163g, PCR1-192g, PCR2-202g
• Nanodrop densities: T2-13.5ng/µl, T1-17.4ng/µl, PCR1-13.5ng/µl, PCR2-17.2ng/µl
• Cells transformed and incubated at 37°C at 9:45am
• T1, T2, RFP, and control removed and plated at 11:15am
• Started uptake and enzyme activity tests with N-methoxy-leu

Lab work:

• Continued performing uptake test for N-methoxy-leu

Lab work:

• Continued performing uptake test for N-methoxy-leu - lysed cells and stored cell lysate
• Performed minipreps and confirmatory digests - No result from digests because no EtBr was in the gel

Lab work:

• Redoing digests from 7/21 and running a new gel
• Received sgRNA from biobasic, prepared and stored the sgRNA at -20°C
• Digested CRISPR RNA insert and Cas9 vector

Lab work:

• Performed digests with BSA1 on the CRISPR vector and insert
• Performed a ligation reaction between the vector and insert

Lab work:

• Realized that the primers used to PCR the LeuS gene were incorrect - Therefore, our T1 and T2 biobricks did not contain the correct DNA sequences

Lab work:

• Continued the PrG uptake test - purification of the cell lysate
• Performed a miniprep for the T1 and T2 terminators (for biobricking)

Lab work:

• Transformed and plated KanR gene from kit plate
• Streaked pS1M27 cells from gel stab to Tet plate

Lab work:

• Re-transformed and plated KanR gene on CAM plates
• Re-streaked pS1M27 plate

Lab work:

• Miniprep performed for pS1M27
• Re-transformed KanR DNA
• Performed a PCR reaction to obtain LeuS gene from genomic DNA

Lab work:

• Confirmatory digest performed on PCR product (LeuS gene)
• Restriction digest for biobrick #1
• Performed ligation reactions between the LeuS gene and the T1 and T2 genes

Lab work:

• Performed a ligation reaction between CRISPR Cas9 genes and sgRNA sequence
• Minipreped ZeoR, KanR, T1, and T2 plasmids
• Confirmatory digests performed for T1 and T2 plasmids
• Performed a ligation reaction with T1 and T2 genes, transformed products

Lab work:

• PCR performed to confirm CRISPR/Cas9 and sgRNA gene ligation

Lab work:

• Digests performed to redo LeuS and terminator ligation

May
S	M	T	W	T	F	S
1	2	3	4	5	6	7
8	9	10	11	12	13	14
15	16	17	18	19	20	21
22	23	24	25	26	27	28
29	30	31

June
S	M	T	W	T	F	S
			1	2	3	4
5	6	7	8	9	10	11
12	13	14	15	16	17	18
19	20	21	22	23	24	25
26	27	28	29	30

July
S	M	T	W	T	F	S
					1	2
3	4	5	6	7	8	9
10	11	12	13	14	15	16
17	18	19	20	21	22	23
24	25	26	27	28	29	30
31

August
S	M	T	W	T	F	S
	1	2	3	4	5	6
7	8	9	10	11	12	13
14	15	16	17	18	19	20
21	22	23	24	25	26	27
28	29	30	31