Parts Documentation

The tables below detail all parts used thus far in our project. Each of these parts can be thought of as components of higher-level assemblies that enable more complex operations within E. coli cells.

In particular:

• Each sgRNA forms the basis of large CRISPR guide libraries for prophage counterselection and recombineering.
• Each RBD and their corresponding homology arms form the basis of the data that the generative phage bank uses to recommend suitable RBDs (and, by extension, phages) to users.

Table 1: Vectors (Backbones) Used for sgRNA and RBD Delivery to E. coli K12

Title	Type	Short Description
pHERD30T	Plasmid	Bacterial expression of Cas9 nuclease, tracrRNA, and crRNA guide.
pCas3cRh	Plasmid	Bacterial expression of IC-CRISPR expression system, with crRNA guide.
pCas3crh-pHERD30T	Plasmid	All-in-one plasmid with the four I-C system genes cloned downstream of the crRNA site. Rhamnose-inducible system.
pCRISPR	Plasmid	A crRNA expression plasmid for targeting a specific sequence.
pETDuet-1	Plasmid	Contains generated RBD with homology arms for the RBD locus in the phage genome, compatible for co-transformation with CRISPR-Cas expression plasmids.

Table 2: sgRNAs Used for RBD Counterselection

sgRNAs for Lambda Phage RBD Counterselection

Number	Title	Type	Short Description
BBa_25SVFR5A	lambda_cas9_2_F	Primer	Forward strand of pCRISPR sgRNA for lambda WT RBP cleavage. pCRISPR is being used as a companion for pCas9 in this workflow.
BBa_25VPB42N	lambda_cas9_2_R	Primer	Reverse strand of pCRISPR sgRNA for lambda WT RBP cleavage. pCRISPR is being used as a companion for pCas9 in this workflow.
BBa_25N96CRU	lambda_1_F	Primer	Forward strand of pCas9 sgRNA for lambda WT RBP cleavage.
BBa_25KMKTGF	lambda_1_R	Primer	Reverse strand of pCas9 sgRNA for lambda WT RBP cleavage.

sgRNAs for HK97 Phage RBD Counterselection

Number	Title	Type	Short Description
BBa_25MKZ7U1	HK97_cas9_2_F	Primer	Forward strand of pCRISPR sgRNA for HK97 WT RBP cleavage. pCRISPR is being used as a companion for pCas9 in this workflow.
BBa_25SQP4Y5	HK97_cas9_2_R	Primer	Reverse strand of pCRISPR sgRNA for HK97 WT RBP cleavage. pCRISPR is being used as a companion for pCas9 in this workflow.
BBa_25WGOFAL	HK97_1_F	Primer	Forward strand of pCas9 sgRNA for HK97 WT RBP cleavage.
BBa_25NSVJAR	HK97_1_R	Primer	Reverse strand of pCas9 sgRNA for HK97 WT RBP cleavage.

sgRNAs for Mu Phage RBD Counterselection

Number	Title	Type	Short Description
BBa_25YZMMM6	1 Mu_Gp49_set1F	Primer	Forward strand of ICCRISPR sgRNA for Mu WT RBP cleavage.
BBa_25KSL9T3	2 Mu_Gp49_set1R	Primer	Reverse strand of ICCRISPR sgRNA for Mu WT RBP cleavage.
BBa_25KWAZBT	3 Mu_Gp49_set2F	Primer	Forward strand of ICCRISPR sgRNA for Mu WT RBP cleavage.
BBa_25EBREV4	4 Mu_Gp49_set2R	Primer	Reverse strand of ICCRISPR sgRNA for Mu WT RBP cleavage.
BBa_25FLG35K	5 Mu_Gp49_set3F	Primer	Forward strand of ICCRISPR sgRNA for Mu WT RBP cleavage.
BBa_25N3F5SM	6 Mu_Gp49_set3R	Primer	Reverse strand of ICCRISPR sgRNA for Mu WT RBP cleavage.
BBa_25G96OQ1	11 Mu_Gp52_set1F	Primer	Forward strand of ICCRISPR sgRNA for Mu GP52 WT RBP cleavage.
BBa_2511CL6U	12 Mu_Gp52_set1R	Primer	Reverse strand of ICCRISPR sgRNA for Mu GP52 WT RBP cleavage.
BBa_25BM1ZPI	13 Mu_Gp52_set2F	Primer	Forward strand of ICCRISPR sgRNA for Mu GP52 WT RBP cleavage.
BBa_25I8S57L	14 Mu_Gp52_set2R	Primer	Reverse strand of ICCRISPR sgRNA for Mu GP52 WT RBP cleavage.
BBa_25ZUKDF1	Mugp49_1_F	Primer	Forward strand of pCas9 sgRNA for Mu GP49 WT RBP cleavage.
BBa_257JMD2V	Mugp49_1_R	Primer	Reverse strand of pCas9 sgRNA for Mu GP49 WT RBP cleavage.
BBa_25QMSD2W	Mugp52_1_F	Primer	Forward strand of pCas9 sgRNA for Mu GP52 WT RBP cleavage.
BBa_25HLLDTM	Mugp52_1_R	Primer	Reverse strand of pCas9 sgRNA for Mu GP52 WT RBP cleavage.
BBa_25ZM7LSH	Mugp49_cas9_2_F	Primer	Forward strand of pCRISPR sgRNA for Mu GP49 WT RBP cleavage. pCRISPR is being used as a companion for pCas9 in this workflow.
BBa_25MVX00K	Mugp49_cas9_2_R	Primer	Reverse strand of pCRISPR sgRNA for Mu GP49 WT RBP cleavage. pCRISPR is being used as a companion for pCas9 in this workflow.
BBa_25YUOYLP	Mugp52_cas9_2_F	Primer	Forward strand of pCRISPR sgRNA for Mu GP52 WT RBP cleavage. pCRISPR is being used as a companion for pCas9 in this workflow.
BBa_25UKZTWS	Mugp52_cas9_2_R	Primer	Reverse strand of pCRISPR sgRNA for Mu GP52 WT RBP cleavage. pCRISPR is being used as a companion for pCas9 in this workflow.

sgRNAs for P2 Phage RBD Counterselection

Number	Title	Type	Short Description
BBa_25VAVDEF	7 P2_sgRNA_set1F	Primer	Forward strand of ICCRISPR sgRNA for P2 WT RBP cleavage.
BBa_25VORT8E	8 P2_sgRNA_set1R	Primer	Reverse strand of ICCRISPR sgRNA for P2 WT RBP cleavage.
BBa_25CWLZ24	9 P2_sgRNA_set2F	Primer	Forward strand of ICCRISPR sgRNA for P2 WT RBP cleavage.
BBa_25O7H6ZZ	10 P2_sgRNA_set2R	Primer	Reverse strand of ICCRISPR sgRNA for P2 WT RBP cleavage.
BBa_25TFRND5	P2_1_F	Primer	Forward strand of pCas9 sgRNA for P2 WT RBP cleavage.
BBa_25IPJ84H	P2_1_R	Primer	Reverse strand of pCas9 sgRNA for P2 WT RBP cleavage.
BBa_25TUTLKJ	P2_cas9_2_F	Primer	Forward strand of pCRISPR sgRNA for P2 WT RBP cleavage. pCRISPR is being used as a companion for pCas9 in this workflow.
BBa_25UFZ72Y	P2_cas9_2_R	Primer	Reverse strand of pCRISPR sgRNA for P2 WT RBP cleavage. pCRISPR is being used as a companion for pCas9 in this workflow.

Table 3: Phage/pETDuet-1 Homology Arm Collection

HAs for P2 Phage Swapping

Number	Title	Type	Short Description
BBa_25DP4ER5	P2 5’_Homology arm	Homologous Region	5’ P2 RBD insert homology arm.
BBa_25CXWX8I	P2 3’_Homology arm	Homologous Region	3’ P2 RBD insert homology arm.

HAs for HK97 Phage Swapping

Number	Title	Type	Short Description
BBa_25FGHDVX	HK97 5’_Homology arm	Homologous Region	5’ HK97 RBD insert homology arm.
BBa_25AVT94A	HK97 3’_Homology arm	Homologous Region	3’ HK97 RBD insert homology arm.

HAs for Lambda Phage Swapping

Number	Title	Type	Short Description
BBa_25ZWOZ2X	Lambda 5’_Homology arm	Homologous Region	5’ Lambda RBD insert homology arm.
BBa_25IP02AV	Lambda 3’_Homology arm	Homologous Region	3’ Lambda RBD insert homology arm.

Table 4: Generated Receptor Binding Domain Collection

Generated RBDs for P2 Phage

Number	Title	Type	Short Description
BBa_25BDXVMP	P2 RBD_2	Protein Domain	Batch 1 generated RBD sequence for P2, same length as the WT P2 RBD (225 bp).
BBa_25YM8GD4	P2 RBD_8	Protein Domain	Batch 1 generated RBD sequence for P2, same length as the WT P2 RBD (225 bp).

Generated RBDs for HK97 Phage

Number	Title	Type	Short Description
BBa_25D782SA	HK97 RBD_2	Protein Domain	Batch 1 generated RBD sequence for HK97, same length as the WT HK97 RBD (330 bp).
BBa_25QHVNNX	HK97 RBD_3	Protein Domain	Batch 1 generated RBD sequence for HK97, same length as the WT HK97 RBD (330 bp).
BBa_252GAZ08	HK97 RBD_5	Protein Domain	Batch 1 generated RBD sequence for HK97, same length as the WT HK97 RBD (330 bp).

Generated RBDs for Lambda Phage

Number	Title	Type	Short Description
BBa_255979JB	Lambda RBD_1	Protein Domain	Batch 1 generated RBD sequence for Lambda, same length as the WT Lambda RBD (399 bp).
BBa_25H4N1YV	Lambda RBD_15	Protein Domain	Batch 1 generated RBD sequence for Lambda, same length as the WT Lambda RBD (399 bp).
BBa_25488QMB	Lambda RBD_16	Protein Domain	Batch 1 generated RBD sequence for Lambda, same length as the WT Lambda RBD (399 bp).
BBa_25VMZMU7	Lambda RBD_19	Protein Domain	Batch 1 generated RBD sequence for Lambda, same length as the WT Lambda RBD (399 bp).
BBa_25NXY6GE	Lambda RBD_22	Protein Domain	Batch 1 generated RBD sequence for Lambda, same length as the WT Lambda RBD (399 bp).

Table 5: Primers Used for sgRNA Cloning, Gibson Assembly, Colony PCR, and RBD Checking

Sequencing Primers

Number	Title	Type	Short Description
BBa_25Q4XK0E	pCas9_seq	Primer	Primer for Sanger sequencing of pCas9 inserts.
BBa_25PPNC6Q	pCRISPR_seq	Primer	Primer for Sanger sequencing of pCas3crh inserts.

RBD Checking Primers

Number	Title	Type	Short Description
BBa_25P5YRMH	P2 RBD_2 R checking primer	Primer	Reverse primer targeting P2 RBD_2. Used to check for P2 RBD_2 in the P2 genome when paired with P2 genome F checking primer.
BBa_25LQLUUJ	P2 RBD_8 R checking primer	Primer	Reverse primer targeting P2 RBD_8. Used to check for P2 RBD_8 in the P2 genome when paired with P2 genome F checking primer.
BBa_25FJ9Z00	P2 wt_RBD R checking primer	Primer	Reverse primer targeting the P2 WT RBD. Used to check for the P2 WT RBD when paired with P2 genome F checking primer.
BBa_25POMAHD	P2 genome F checking primer	Primer	Forward primer targeting the P2 genome upstream of the sequence homologous to P2 5’_Homology arm. Used to check for an RBD in the P2 genome when paired with a reverse primer targeting a specific RBD.
BBa_2551JLWJ	HK97 RBD_2 R checking primer	Primer	Reverse primer targeting HK97 RBD_2. Used to check for HK97 RBD_2 in the HK97 genome when paired with HK97 genome F checking primer.
BBa_25XRXVMM	HK97 RBD_3 R checking primer	Primer	Reverse primer targeting HK97 RBD_3. Used to check for HK97 RBD_3 in the HK97 genome when paired with HK97 genome F checking primer.
BBa_25DRS1ZS	HK97 RBD_5 R checking primer	Primer	Reverse primer targeting HK97 RBD_5. Used to check for HK97 RBD_5 in the HK97 genome when paired with HK97 genome F checking primer.
BBa_25V3M31Z	HK97 wt_RBD R checking primer	Primer	Reverse primer targeting the HK97 WT RBD. Used to check for HK97 WT RBD in the HK97 genome when paired with HK97 genome F checking primer.
BBa_25K1LHYL	HK97 genome F checking primer	Primer	Forward primer targeting the HK97 genome upstream of the sequence homologous to HK97 5’_Homology arm. Used to check for an RBD in the HK97 genome when paired with a reverse primer.
BBa_259S1IZM	Lambda RBD_1 R checking primer	Primer	Reverse primer targeting Lambda RBD_1. Used to check for Lambda RBD_1 when paired with Lambda genome F checking primer.
BBa_251VZPHK	Lambda RBD_15 R checking primer	Primer	Reverse primer targeting Lambda RBD_15. Used to check for Lambda RBD_15 when paired with Lambda genome F checking primer.
BBa_25TWJ1DJ	Lambda RBD_16 R checking primer	Primer	Reverse primer targeting Lambda RBD_16. Used to check for Lambda RBD_16 when paired with Lambda genome F checking primer.
BBa_25UQ5F7F	Lambda RBD_19 R checking primer	Primer	Reverse primer targeting Lambda RBD_19. Used to check for Lambda RBD_19 when paired with Lambda genome F checking primer.
BBa_25Z9NGEB	Lambda RBD_22 R checking primer	Primer	Reverse primer targeting Lambda RBD_22. Used to check for Lambda RBD_22 when paired with Lambda genome F checking primer.
BBa_25NGDB8W	Lambda wt_RBD R checking primer	Primer	Reverse primer targeting the Lambda WT RBD. Used to check for Lambda WT RBD when paired with Lambda genome F checking primer.
BBa_25MW1CXI	Lambda genome F checking primer	Primer	Forward primer targeting the Lambda genome upstream of the sequence homologous to Lambda 5’_Homology arm. Used to check for an RBD in the Lambda genome when paired with a reverse primer.